CN115678913A - 表观遗传因子在真核细胞中优化基因编辑工具的应用 - Google Patents

Abstract

本发明公开了表观遗传因子在真核细胞中优化基因编辑工具的应用。本发明所要保护的重组基因编辑系统为在基因编辑系统的基础上改造得到的，所述重组基因编辑系统表达融合蛋白，所述融合蛋白含有序列特异性结合蛋白、诱导基因组修饰因子和表观遗传因子；所述重组基因编辑系统的基因编辑效率高于所述基因编辑系统。所述重组基因编辑系统可为载体、mRNA或DNA分子。所述重组基因表达系统、表观遗传因子、融合蛋白和/或融合蛋白相关的生物材料可应用于优化基因编辑，在实际生产中，可提高基因编辑效率，实现新的编辑类型和建立新兴的基因编辑工具。

Description

表观遗传因子在真核细胞中优化基因编辑工具的应用

技术领域

本发明涉及生物技术领域，具体涉及表观遗传因子在真核细胞中优化基因编辑工具的方式及其应用。

背景技术

基因编辑系统是指一类特异性靶向基因组，并诱导基因组突变的蛋白系统。现有的基因编辑系统主要由序列特异性结合蛋白(Cas9蛋白、锌指核酸酶、转录激活物样效应核酸酶等)和诱导基因组修饰因子(脱氨酶、转座酶、逆转录酶等)组成，可诱导基因组的突变、插入、缺失，为基因突变导致的疾病治疗带来曙光。目前，这类基因编辑系统的优化工作主要集中在基因组结合蛋白及诱导因子的进化，例如靶向基因组蛋白从锌指核酸酶向Cas9蛋白的转变，此外，诱导基因组修饰因子也从最初的空白转变为脱氨酶、转座酶等。如碱基编辑系统，CBE基因编辑系统为胞嘧啶碱基编辑系统，CBE中的胞苷脱氨酶可直接将目标胞苷转化为尿苷，随后在尿嘧啶糖基化酶抑制剂和DNA复制的作用下将尿嘧啶转化为胸腺嘧啶，可在不对DNA双链进行切割的条件下完成C-T碱基的转换；GBE碱基编辑系统会在尿嘧啶羰基化酶的作用下，切除尿苷，随后在细胞自身的DNA修复机制下，实现C-G碱基的转换。然而，现阶段少有研究探索基因组外部环境的变化对基因编辑系统的功能影响，尤其是在真核细胞中。真核细胞中，基因组的基本结构为DNA和组蛋白缠绕的核小体，这种紧密的染色质结构为靶向基因组蛋白与DNA序列的结合设置巨大的障碍，很大程度影响基因编辑系统的效率。

此外，真核细胞的染色质环境不仅包括DNA和组蛋白的结合情况，同时也包括组蛋白尾端氨基酸及DNA自身的化学修饰，这二者共同组成了复杂的染色质环境。可以预见，真核细胞复杂的染色质环境必将对基因编辑系统的功能产生巨大的影响。

目前，表观遗传涉及的因子包括染色质重塑因子、组蛋白修饰因子、DNA及RNA修饰因子、miRNA和lncRNA等。染色质重塑因子是一类可与组蛋白相互作用，从而诱导染色质环境改变的复合物；组蛋白修饰因子是组蛋白翻译后修饰的主要蛋白，包括甲基化、乙酰化、泛素化等修饰因子，对基因的转录调控、DNA链的断裂修复等过程有着重要影响；DNA及RNA修饰是发生在DNA及RNA自身的化学修饰，常见的有胞嘧啶的甲基化、腺嘌呤的甲基化修饰，这些修饰同样影响着基因转录调控及DNA修复进程。

发明内容

本发明所要解决的技术问题是如何优化基因编辑系统和/或如何使用表观遗传因子优化基因编辑系统。

为了解决上述技术问题，本发明首先提供了重组基因编辑系统。所述重组基因编辑系统可在基因编辑系统的基础上改造得到的。所述重组基因编辑系统可表达融合蛋白。所述融合蛋白可含有序列特异性结合蛋白、诱导基因组修饰因子和表观遗传因子。所述重组基因编辑系统的基因编辑效率高于所述基因编辑系统。

上文所述重组基因编辑系统可为载体、mRNA或DNA分子。

所述基因编辑系统可含有序列特异性结合蛋白的编码基因和诱导基因组修饰因子的编码基因。所述基因编辑系统可表达含有序列特异性结合蛋白和诱导基因组修饰因子的融合蛋白。

上文所述重组基因编辑系统中，所述基因编辑系统可为碱基编辑系统。所述碱基编辑系统可为CBE碱基编辑系统或GBE碱基编辑系统。所述CBE碱基编辑系统可为BE4max碱基编辑系统。所述基因编辑系统还可为其他基因编辑系统，如先导编辑器或基于转座酶的基因编辑系统等。上文所述重组基因编辑系统可为重组碱基编辑系统。

上文所述重组基因编辑系统中，所述序列特异性结合蛋白可为Cas9蛋白。所述诱导基因组修饰因子可为脱氨酶。

上文所述重组碱基编辑系统中，所述序列特异性结合蛋白还可为锌指核酸酶或转录激活物样效应核酸酶等。所述诱导基因组修饰因子还可为转座酶或逆转录酶等。

上文所述重组基因编辑系统中，所述表观遗传因子可为染色质重塑因子、组蛋白修饰因子和/或RNA修饰因子。

上文所述重组基因编辑系统中，所述基因编辑系统可为CBE碱基编辑系统或GBE碱基编辑系统。所述脱氨酶可为胞苷脱氨酶。所述Cas9蛋白可为nCas9。

上文所述重组基因编辑系统中，所述Cas9蛋白还可为其他Cas9蛋白。所述脱氨酶还可为其他脱氨酶。所述融合蛋白还可含有其他蛋白。所述其他蛋白可为尿嘧啶糖基化酶抑制剂蛋白(UGI)和/或尿嘧啶糖基化酶(UDG)。所述尿嘧啶糖基化酶抑制剂蛋白的氨基酸序列可为序列表中序列1的第2006-2088位。所述尿嘧啶糖基化酶的氨基酸序列可为序列表中序列3的第1993-2224位。

上文所述重组基因编辑系统中，所述胞苷脱氨酶的氨基酸序列可为序列表中序列1的第20-247位。所述nCas9蛋白的氨基酸序列可为序列表中序列1的第629-1995位。

上文所述重组基因编辑系统中，所述染色质重塑因子可为SOX2。所述组蛋白修饰因子可为SETD2。所述RNA修饰因子可为METTL3。

所述SOX2可为A1)、A2)、A3)、A4)或A5)中的任一种：

A1)氨基酸序列为序列表中序列1的第280-596位。

A2)由编码序列是序列表中序列6的第838-1053位的核苷酸序列编码的蛋白质。

A3)由编码序列是序列表中序列7的第838-1188位的核苷酸序列编码的蛋白质。

A4)将A1)、A2)或A3)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)、A2)或A3)衍生的或与A1)、A2)或A3)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质。

A5)在A1)、A2)、A3)或A4)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

上文所述重组基因编辑系统中，所述SETD2可为B1)、B2)或B3)中的任一种：

B1)由编码序列是序列表中序列9的第25-915位的核苷酸序列编码的蛋白质。所述SETD2的氨基酸序列由297个氨基酸残基组成。

B2)将B1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由B1)衍生的或与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质。

B3)在B1)或B2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

上文所述重组基因编辑系统中，所述METTL3可为C1)、C2)或C3)中的任一种：

C1)由编码序列是序列表中序列10的第25-1761位的核苷酸序列编码的蛋白质。所述METTL3的氨基酸序列由579个氨基酸残基组成。

C2)将C1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由C1)衍生的或与C1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质。

C3)在C1)或C2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

上文所述重组基因编辑系统中，所述融合蛋白可为D1)、D2)、D3)、D4)、D5)、D6)中的任一种：

D1)氨基酸序列为序列表中序列1的蛋白质。

D2)氨基酸序列为序列表中序列3的蛋白质。

D3)由编码序列是序列表中序列6的第1-5871位的核苷酸序列编码的蛋白质。所述融合蛋白的氨基酸序列由1957个氨基酸残基组成。

D4)由编码序列是序列表中序列7的第1-6006位的核苷酸序列编码的蛋白质。所述融合蛋白的氨基酸序列由2002个氨基酸残基组成。

D5)由编码序列是序列表中序列8的第1-6072位的核苷酸序列编码的蛋白质。所述融合蛋白的氨基酸序列由2024个氨基酸残基组成。

D6)由编码序列是序列表中序列9的第1-6612位的核苷酸序列编码的蛋白质。所述融合蛋白的氨基酸序列由2224个氨基酸残基组成。

D7)由编码序列是序列表中序列10的第1-7458位的核苷酸序列编码的蛋白质。所述融合蛋白的氨基酸序列由2486个氨基酸残基组成。

D8)将D1)、D2)、D3)、D4)、D5)、D6)或D7)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由D1)、D2)、D3)、D4)、D5)、D6或D7)衍生的或与D1)、D2)、D3)、D4)、D5)、D6)或D7)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质。

D9)在D1)、D2)、D3)、D4)、D5)、D6)、D7)或D8)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

所述标签蛋白(protein-tag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签蛋白可为Flag标签蛋白、His标签蛋白、MBP标签蛋白、HA标签蛋白、myc标签蛋白、GST标签蛋白和/或SUMO标签蛋白等。

上文所述的融合蛋白或其相关的生物材料也属于本发明的保护范围。所述生物材料可为下述任一种：

E1)编码上文所述融合蛋白质的核酸分子。

E2)含有E1)所述核酸分子的表达盒。

E3)含有E1)所述核酸分子的重组载体、或含有E2)所述表达盒的重组载体。

E4)含有E1)所述核酸分子的重组微生物、或含有E2)所述表达盒的重组微生物、或含有E3)所述重组载体的重组微生物。

E5)含有E1)所述核酸分子的转基因植物细胞系、或含有E2)所述表达盒的转基因细胞系、或含有E3)所述重组载体的转基因细胞系。

E6)含有E1)所述核酸分子的转基因植物组织、或含有E2)所述表达盒的转基因组织、或含有E3)所述重组载体的转基因组织。

E7)含有E1)所述核酸分子的转基因动物器官、或含有E2)所述表达盒的转基因动物器官、或含有E3)所述重组载体的转基因动物器官。

上文所述生物材料中，B1)所述核酸分子可为下述任一种：

E11)编码序列是序列表中序列2的DNA分子。

E12)编码序列是序列表中序列4的DNA分子。

E13)编码序列是序列表中序列6的DNA分子。

E14)编码序列是序列表中序列7的DNA分子。

E15)编码序列是序列表中序列8的DNA分子。

E16)编码序列是序列表中序列9的DNA分子。

E17)编码序列是序列表中序列10的DNA分子。

为了解决上述技术问题本发明还提供了上文所述的表观遗传因子在提高基因编辑系统的基因编辑效率中的应用。

为了解决上述技术问题本发明还提供了上文所述的表观遗传因子和/或上文所述的融合蛋白和/或其相关的生物材料在基因编辑中的应用。

所述应用的目的可以是疾病诊断目的、疾病预后目的和/或疾病治疗目的，它们的目的也可以是非疾病诊断目的、非疾病预后目的和非疾病治疗目的；所述应用的直接目的可以是获取疾病诊断结果、疾病预后结果和/或疾病治疗结果的中间结果的信息，所述应用的直接目的可以是非疾病诊断目的、非疾病预后目的和/或非疾病治疗目的。

本发明将表观遗传因子的编码基因导入CBE和GBE碱基编辑系统得到表达含有表观遗传因子的融合蛋白的重组碱基编辑系统，并将该重组碱基编辑系统质粒和靶向于特定基因的gRNA质粒共转染HEK293T细胞进行碱基编辑编辑效率分析，结果发现，相比较于CBE和GBE碱基编辑系统，重组碱基编辑系统可在不同程度上提高碱基的编辑范围和碱基的编辑效率。本发明所提供的重组基因编辑系统和/或表观遗传因子在优化基因编辑系统中的应用在实际生产中，可提高基因编辑效率，实现新的编辑效果和建立新兴的基因编辑工具。

附图说明

图1为先锋因子融合碱基编辑系统图。

图2为先锋因子蛋白融合编辑器编辑结果图。

图3为细胞转染及筛选示意图。

图4为HEK293T(293T)细胞中融合先锋因子SOX2蛋白对CBE(A)和GBE(B)编辑结果的影响。(A)的纵坐标为碱基C-T转换的效率，横坐标为胞嘧啶在前间隔基序框的位置；(B)的纵坐标为碱基C-G转换的效率。

图5为MYC终止突变靶位点C的编辑效率(A)和诱导终致突变的C11编辑效率(B)。(A)的纵坐标为碱基C-T转换的效率；(B)的纵坐标为11号位胞嘧啶的C-T转换效率。

图6为SOX2结构域拆分对CBE(A)和GBE(B)编辑效率的影响。(A)的纵坐标为碱基C-T转换的效率，横坐标为胞嘧啶在前间隔基序框的位置；(B)的纵坐标为碱基C-G转换的效率，横坐标为GBE和基于SOX2蛋白不同结构域构建的GBE。

图7为SETD2和METTL3蛋白对GBE编辑效率的影响。纵坐标为碱基C-G转换的效率。

具体实施方式

下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。

下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

本发明实施例中BE4max碱基编辑系统表达质粒来源于Addgene(#112093)。GBE碱基编辑系统表达质粒(APOBEC-nCas9-Ung)和gRNA表达质粒骨架(RNF2 sgRNA)为实验室保存(相关文献：Zhao D,Li J,Li S,et al.New base editors change C to A in bacteriaand C to G in mammalian cells[J].Nature Biotechnology,2021,39(1).，公众可从申请人处获得，仅用于重复本发明)。HEK293T细胞(293T)和宫颈癌细胞(HeLa)源于北京大学医学部尚永丰教授赠送(相关文献：Yang C,Wu J,Liu X,et al.Circadian Rhythm IsDisrupted by ZNF704 in Breast Carcinogenesis[J].Cancer Research,2020,80(19):canres.0493.2020.)。

实施例1：筛选优化碱基编辑系统的一类表观遗传因子-先锋因子蛋白

1.测试先锋因子

先锋因子是染色质重塑因子中的一类，筛选标准包括：①先锋因子功能已被文献报道；②基因编码序列长度在500bp-2000bp之间；③先锋因子功能结构域划分清晰。

通过筛选拟进行如下先锋因子蛋白PAX7(NG_023262)，PBX1(NG_028246)，FOXA1(NG_033028)，SOX2(NG_009080，氨基酸序列为序列表中序列1的第280-596位)的验证。

2.构建含有先锋因子蛋白的重组基因编辑系统

2.1引物设计和PCR扩增

通过RNA提取试剂盒提取293T(HEK293T)细胞RNA，并以提取的RNA为模板反转录获取cDNA文库。

根据步骤1筛选得到的先锋因子基因序列设计上下游引物(表1)，以获取的cDNA文库为模板进行PCR扩增，分别获得筛选先锋因子PAX7，PBX1，FOXA1，SOX2的基因序列(核苷酸序列为序列表中序列2的第838-1788位)。

表1.PCR扩增引物列表

2.2构建不同先锋因子排列组合的编辑系统表达质粒

BE4max碱基编辑系统(一种CBE碱基编辑系统)能表达融合蛋白APOBEC1-nCas9-2xUGI；GBE碱基编辑系统能表达融合蛋白APOBEC1-nCas9-UNG。

其中APOBEC1为胞苷脱氨酶(氨基酸序列为序列表中序列1的第20-247位)，nCas9为Cas9蛋白(氨基酸序列为序列表中序列1的第629-1995位)，UGI为尿嘧啶糖基化酶抑制蛋白(氨基酸序列为序列表中序列1的第2006-2088位)，UNG为尿嘧啶糖基化酶(氨基酸序列为序列表中序列3的第1993-2224位)。

以BE4max、GBE碱基编辑系统为基础，使用无缝克隆试剂盒(碧云天，货号：D7010S)通过基因重组方法分别将步骤2.1中扩增得到的四种不同先锋因子的基因序列分别整合至BE4max碱基编辑系统质粒和GBE碱基编辑系统质粒中。

同时考虑到先锋因子基因序列插入到碱基编辑系统质粒的位置不同，导致得到的重组碱基编辑系统表达的融合蛋白中先锋因子蛋白与脱氨酶、Cas蛋白的排列位置影响编辑系统的编辑效果，本发明通过无缝克隆试剂盒分别将PAX7，PBX1，FOXA1，SOX2四种先锋因子基因序列插入到CBE和GBE碱基编辑系统质粒上APOBEC1基因编码序列和nCas9基因蛋白编码序列的不同位置，分别得到先锋因子蛋白表达在如图1所示的融合蛋白APOBEC1-nCas9-2xUGI或APOBEC1-nCas9-UNG的APOBEC1的氨基端位置(NH₃)、中间位置(Middle，即APOBEC1和nCas9中间位置)和nCas9的羧基端位置(COOH)的新的融合蛋白，以验证编辑效果。

实验结果证实，将先锋因子蛋白SOX2的编码序列插入到BE4max碱基编辑系统质粒中间位置得到的重组碱基编辑系统SoxM-CBE(图2中A的SOX2-middle-CBE)的碱基编辑效率高于BE4max碱基编辑系统(图2中A的BE4max)和将其他三种先锋因子蛋白的编码序列插入到BE4max碱基编辑系统质粒中间位置得到的重组碱基编辑系统(图2中A的PAX7-middle-CBE、PBX1-middle-CBE和FOXA1-middle-CBE)；将先锋因子蛋白SOX2的编码序列插入到GBE碱基编辑系统质粒氨基端位置得到的重组碱基编辑系统SoxN-GBE(SOX2-NH3-GBE，图2中B的SOX2-GBE(N))的碱基编辑效率在RP11位点高于GBE碱基编辑系统(图2中B的GBE)和将其他三种先锋因子蛋白的编码序列插入到GBE碱基编辑系统质粒氨基端位置得到的重组碱基编辑系统(图2中B的PAX7-GBE(N)、PBX1-GBE(N)和FOXA1-GBE(N))。

基于BE4max碱基编辑系统得到的SoxM-CBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列2的重组基因APOBEC1-SOX2-nCas9-2xUGI，能够表达氨基酸序列为序列表中序列1的融合蛋白APOBEC1-SOX2-nCas9-2xUGI。

融合蛋白APOBEC1-SOX2-nCas9-2xUGI从N端到C端依次为胞苷脱氨酶1(APOBEC1)、先锋因子SOX2、Cas9蛋白(nCas9)，尿嘧啶糖基化酶抑制蛋白(UGI)。序列表中序列2的第58-741位核苷酸为APOBEC1基因的编码序列，序列2的第1885-5985位核苷酸为nCas9基因的编码序列，序列2的第6016-6264位核苷酸为UGI基因的编码序列。

基于GBE碱基编辑系统得到的SoxN-GBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列4的重组基因SOX2-APOBEC1-nCas9-UNG，能够表达氨基酸序列为序列表中序列3的融合蛋白SOX2-APOBEC1-nCas9-UNG。

融合蛋白SOX2-APOBEC1-nCas9-UNG从N端到C端依次为先锋因子SOX2、胞苷脱氨酶1(APOBEC1)、Cas9蛋白(nCas9)，尿嘧啶糖基化酶(UNG)。序列表中序列4的第5977-6672位核苷酸为UNG基因的编码序列。

实施例2：293T细胞中验证先锋因子对碱基编辑系统的优化效果

1.gRNA重组表达质粒的构建

依据GBE和BE4max碱基编辑系统的特性，构建gRNA表达质粒，每种碱基编辑系统构建gRNA表达质粒各10个(表2)。

表2.sgRNA的克隆和深度测序引物

具体为以RNF2 sgRNA序列为模板，设计上下游引物(靶基因组位点名称及对应扩增引物见表2)，引物退火形成gRNA的编码双链DNA，应用goldengate方法(BsaI酶，Thermo)将得到的gRNA的编码双链DNA与gRNA表达质粒骨架(RNF2 sgRNA)连接，共得到18种gRNA表达质粒。

2.重组质粒转染293T细胞

将实施例1中得到的重组碱基编辑系统SoxM-CBE或SoxN-GBE的质粒和实施例2步骤1中得到的18种gRNA重组表达质粒分别转化大肠杆菌Trans5α(Transgene公司)进行扩增，通过质粒提取试剂盒(天根)分别获取重组碱基编辑系统质粒和gRNA表达质粒。

传代生长融合度达90％的293T细胞于细胞培养24孔板中，第二天进行质粒转染实验，实验中分别转染SoxM-CBE或SoxN-GBE质粒600ng、gRNA表达质粒每种300ng(SoxN-GBE+gRNA，SoxM-CBE+gRNA)，并加入转染试剂PEI(Polysciences,美国)(转染质粒和PEI的比例为1μg：3μL)和Opti-MEM培养基(Gibco)(转染质粒和培养基的比例1μg：100μL)，充分混匀后静置15min，加入至待转染的293T细胞中转染24h；转染后24h将转染细胞更换含有嘌呤霉素(Sigma)(比例为1：2500)的培养基(Gibco)继续培养，共培养6天，每两天更换1次培养基(图3)。

3.融合先锋因子SOX2的重组碱基编辑系统编辑效率的验证

步骤2中细胞转染后6天收集转染细胞，PBS清洗一遍后，加入适量细胞裂解液，提取细胞基因组DNA，以转染细胞的基因组DNA为模板，PCR扩增获取目的片段，并通过纯化试剂盒回收目的片段，进行深度测序分析；Linux系统下通过CRISPResso2软件分析深度测序数据，统计并比较不同先锋因子融合碱基编辑系统的编辑效率、编辑框(图4)，结果显示，BE4max碱基高效编辑范围为4-9位，经过优化后的重组碱基编辑系统SoxM-CBE的高效碱基编辑范围为5-16位，SoxM-CBE表现出更广的编辑范围(图4中A)；GBE碱基编辑系统的平均编辑效率为13.73％，经过优化后的重组碱基编辑系统SoxN-GBE的平均编辑效率为28.32％，SoxN-GBE表现出更高的编辑效率(图4中B)。

实施例3：宫颈癌细胞中应用SoxM-CBE系统诱导原癌基因MYC的终止突变

1.SoxM-CBE碱基编辑系统质粒转染宫颈癌癌症细胞

传代培养宫颈癌癌症细胞HeLa，当传代生长融合度达90％时，将细胞传代分至24孔板中，第二天进行质粒转染实验，实验中分别转染实施例1中得到的重组碱基编辑系统质粒(SoxM-CBE 600ng、实施例2步骤1中得到1种gRNA重组质粒(gRNA靶位点序列：5’-CACGGCCGACCAGCTGGAGA-3’，靶基因为MYC基因(表2中MYC-site))300ng，并加入转染试剂PEI(转染质粒与PEI的比例为1μg：3μL)和Opti-MEM培养基(转染质粒与培养基的比例1μg：100μL)，充分混匀后静置15min，加入至待转染的细胞中；转染后24h更换培养基为嘌呤霉素(比例为1：2500)筛选培养基继续培养，共培养6天，每两天更换1次筛选培养基。

2.重组碱基编辑系统的编辑效率分析

步骤1中收集的转染细胞，PBS清洗一遍后，加入适量细胞裂解液，提取细胞基因组DNA，设计MYC基因靶位点DNA序列上下游引物(F:5’-CCCTCCTACGTTGCGGTCA-3’，R:5’-CGAGAAGCCGCTCCACAT-3’)，并以提取的基因组DNA为模板，PCR扩增获取PCR产物(序列表中序列5)，通过纯化试剂盒回收PCR产物片段，进行深度测序分析，分析MYC基因靶位点DNA序列终止密码子诱导编辑效率(图5)，结果显示，相比于对照BE4max碱基编辑系统，重组碱基编辑系统SoxM-CBE在编辑框第11位胞嘧啶C11的位置表现出近30％的C-T的碱基转换效率。

综上，本发明研究证实当先锋因子SOX2插入GBE碱基编辑系统脱氨酶的氨基端(SoxN-GBE：SOX2-APOBEC1-nCas9-UNG)和CBE碱基编辑系统脱氨酶羧基端(SoxM-BE4max：APOBEC1-SOX2-nCas9-UGI)得到的重组碱基编辑系统，能分别促进GBE和CBE碱基编辑系统编辑效率提升和编辑范围拓展。

实施例4：探索先锋因子SOX2蛋白功能结构域对碱基编辑的影响

1.拆分SOX2功能结构域，构建基于SOX2蛋白功能结构域的重组碱基编辑系统

SOX2蛋白主要包括三个功能结构域(图6)，包括HMG(High mobility group)、RBD(RNA binding domain)、SAD(SOX2 activation domain)。以实施例1步骤2.1中的cDNA为模板，通过PCR(引物见表1)扩增SOX2蛋白三个功能结构域的DNA片段，随后通过无缝克隆试剂盒连接至BE4max碱基编辑系统质粒和GBE碱基编辑系统质粒，筛选获得基于三个功能结构域的6种重组碱基编辑系统，包括HMG-Middle-CBE、RBD-Middle-CBE、SAD-Middle-CBE、HMG-NH3-GBE、RBD-NH3-GBE、SAD-NH3-GBE。

2.细胞水平验证融合不同结构域的碱基编辑系统效果

将步骤1得到的6种重组碱基编辑系统质粒600ng、实施例2步骤1中得到1种gRNA重组质粒(靶基因为FANCF，靶位点序列和扩增引物见表2)300ng按照实施例3步骤1的转染方法共转染至293T细胞，并检测碱基编辑效果。结果显示，相比较于CBE碱基编辑系统，HMG-Middle-CBE(图6中A的HmgM-CBE)和SAD-Middle-CBE(图6中A的SadM-CBE)重组碱基编辑系统在前间隔基序框靠后位置的胞嘧啶均表现出明显的效率提升(编辑范围扩展)；相比较于GBE碱基编辑系统SAD-NH3-GBE(图6中B的SadN-GBE)重组碱基编辑系统对VISTA序列编辑框内6号位胞嘧啶编辑效果有明显的提升(图6中B)。

基于CBE碱基编辑系统得到的HmgM-CBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列6的重组基因APOBEC1-HMG-nCas9-2xUGI，能够表达融合蛋白APOBEC1-HMG-nCas9-2xUGI，融合蛋白APOBEC1-HMG-nCas9-2xUGI的氨基酸序列由1957个氨基酸残基组成，为由编码序列是序列表中序列6的第1-5871位的核苷酸序列编码的蛋白质。

融合蛋白APOBEC1-HMG-nCas9-2xUGI从N端到C端依次为胞苷脱氨酶1(APOBEC1)、先锋因子SOX2的HMG结构域、Cas9蛋白(nCas9)，尿嘧啶糖基化酶抑制蛋白(UGI)。HMG结构域的氨基酸序列由72个氨基酸残基组成，为由编码序列是序列表中序列6的第838-1053位的核苷酸序列编码的蛋白质。。

基于CBE碱基编辑系统得到的SadM-CBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列7的重组基因APOBEC1-SAD-nCas9-2xUGI，能够表达融合蛋白APOBEC1-SAD-nCas9-2xUGI，融合蛋白APOBEC1-SAD-nCas9-2xUGI的氨基酸序列由2002个氨基酸残基组成，为由编码序列是序列表中序列7的第1-6006位的核苷酸序列编码的蛋白质。

融合蛋白APOBEC1-SAD-nCas9-2xUGI从N端到C端依次为胞苷脱氨酶1(APOBEC1)、先锋因子SOX2的SAD结构域、Cas9蛋白(nCas9)，尿嘧啶糖基化酶抑制蛋白(UGI)。SAD结构域的氨基酸序列由117个氨基酸残基组成，为由编码序列是序列表中序列7的第838-1188位的核苷酸序列编码的蛋白质。

基于GBE碱基编辑系统得到的SadN-GBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列8的重组基因SAD-APOBEC1-nCas9-UNG，能够表达融合蛋白SAD-APOBEC1-nCas9-UNG，融合蛋白SAD-APOBEC1-nCas9-UNG的氨基酸序列由2024个氨基酸残基组成，为由编码序列是序列表中序列8的第1-6072位的核苷酸序列编码的蛋白质。

融合蛋白SAD-APOBEC1-nCas9-UNG从N端到C端依次为先锋因子SOX2的SAD结构域、胞苷脱氨酶(APOBEC1)、Cas9蛋白(nCas9)，尿嘧啶糖基化酶(UNG)

实施例5：表观遗传因子SETD2及METTL3对GBE编辑系统的影响

1.构建基于SETD2和METTL3蛋白的GBE重组碱基编辑系统

检索文献筛选表观遗传因子组蛋白甲基转移酶SETD2和RNA甲基转移酶METTL3蛋白进行碱基编辑系统优化，构建GBE重组碱基编辑系统，以293T的cDNA为模板并通过PCR扩增SETD2基因核心酶催化结构域片段(序列表中序列9的第25-915位核苷酸)和METTL3基因片段(序列表中序列10的第25-1761位核苷酸)，扩增引物见表1，随后分别通过无缝克隆试剂盒连接至GBE碱基编辑系统质粒，筛选获得新型的重组碱基编辑系统，包括SETD2-NH3-GBE、和METTL3-NH3-GBE。

基于GBE碱基编辑系统得到的SETD2-NH3-GBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列9的重组基因SETD2-APOBEC1-nCas9-UNG，能够表达融合蛋白SETD2-APOBEC1-nCas9-UNG，融合蛋白SETD2-APOBEC1-nCas9-UNG的氨基酸序列由2224个氨基酸残基组成，为由编码序列是序列表中序列9的第1-6612位的核苷酸序列编码的蛋白质。

融合蛋白SETD2-APOBEC1-nCas9-UNG从N端到C端依次为组蛋白甲基转移酶SETD2、胞苷脱氨酶(APOBEC1)、Cas9蛋白(nCas9)，尿嘧啶糖基化酶(UNG)。SETD2的氨基酸序列由297个氨基酸残基组成，为由编码序列是序列表中序列9的第25-915位的核苷酸序列编码的蛋白质。

基于GBE碱基编辑系统得到的METTL3-NH3-GBE重组碱基编辑系统质粒含有核苷酸序列为序列表中序列10的重组基因METTL3-APOBEC1-nCas9-UNG，能够表达融合蛋白METTL3-APOBEC1-nCas9-UNG，融合蛋白METTL3-APOBEC1-nCas9-UNG的氨基酸序列由2486个氨基酸残基组成，为由编码序列是序列表中序列10的第1-7458位的核苷酸序列编码的蛋白质。

融合蛋白METTL3-APOBEC1-nCas9-UNG从N端到C端依次为RNA甲基转移酶METTL3、胞苷脱氨酶1(APOBEC1)、Cas9蛋白(nCas9)，尿嘧啶糖基化酶(UNG)。METTL3的氨基酸序列由579个氨基酸残基组成，为由编码序列是序列表中序列10的第25-1761位的核苷酸序列编码的蛋白质。

2.细胞水平验证SETD2-NH3-GBE和METTL3-NH3-GBE重组碱基编辑系统效果

分别将SETD2-NH3-GBE和METTL3-NH3-GBE重组碱基编辑系统和实施例2步骤1中得到1种gRNA表达质粒(靶基因为VISTA，靶位点序列及扩增引物见表2)按照实施例3步骤1的方法共转染至293T细胞，并检测靶基因中编辑框内6号位胞嘧啶的碱基编辑效果，结果显示SETD2-NH3-GBE和METTL3-NH3-GBE重组碱基编辑系统相对于GBE编辑系统的编辑效果有明显的提升(图7)。

以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

序列表

<110> 中国科学院天津工业生物技术研究所

<120> 表观遗传因子在真核细胞中优化基因编辑工具的应用

<130> GNCSQ212224

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> 人工序列(Artificial Sequence)

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Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe

210 215 220

Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile

225 230 235 240

Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser

245 250 255

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

260 265 270

Gly Gly Ser Ser Gly Gly Ser Met Tyr Asn Met Met Glu Thr Glu Leu

275 280 285

Lys Pro Pro Gly Pro Gln Gln Thr Ser Gly Gly Gly Gly Gly Asn Ser

290 295 300

Thr Ala Ala Ala Ala Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val

305 310 315 320

Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg

325 330 335

Lys Met Ala Gln Glu Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys

340 345 350

Arg Leu Gly Ala Glu Trp Lys Leu Leu Ser Glu Thr Glu Lys Arg Pro

355 360 365

Phe Ile Asp Glu Ala Lys Arg Leu Arg Ala Leu His Met Lys Glu His

370 375 380

Pro Asp Tyr Lys Tyr Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys

385 390 395 400

Lys Asp Lys Tyr Thr Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn

405 410 415

Ser Met Ala Ser Gly Val Gly Val Gly Ala Gly Leu Gly Ala Gly Val

420 425 430

Asn Gln Arg Met Asp Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly

435 440 445

Ser Tyr Ser Met Met Gln Asp Gln Leu Gly Tyr Pro Gln His Pro Gly

450 455 460

Leu Asn Ala His Gly Ala Ala Gln Met Gln Pro Met His Arg Tyr Asp

465 470 475 480

Val Ser Ala Leu Gln Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met

485 490 495

Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro

500 505 510

Gly Met Ala Leu Gly Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser

515 520 525

Ser Ser Pro Pro Val Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys

530 535 540

Gln Ala Gly Asp Leu Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala

545 550 555 560

Glu Val Pro Glu Pro Ala Ala Pro Ser Arg Leu His Met Ser Gln His

565 570 575

Tyr Gln Ser Gly Pro Val Pro Gly Thr Ala Ile Asn Gly Thr Leu Pro

580 585 590

Leu Ser His Met Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu

595 600 605

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser

610 615 620

Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr

625 630 635 640

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

645 650 655

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

660 665 670

Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala

675 680 685

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

690 695 700

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

705 710 715 720

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

725 730 735

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

740 745 750

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

755 760 765

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

770 775 780

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

785 790 795 800

Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val

805 810 815

Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly

820 825 830

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

835 840 845

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

850 855 860

Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys

865 870 875 880

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

885 890 895

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

900 905 910

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

915 920 925

Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu

930 935 940

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

945 950 955 960

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

965 970 975

Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly

980 985 990

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

995 1000 1005

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

1010 1015 1020

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

1025 1030 1035 1040

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

1045 1050 1055

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

1060 1065 1070

Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg

1075 1080 1085

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

1090 1095 1100

Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu

1105 1110 1115 1120

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

1125 1130 1135

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

1140 1145 1150

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

1155 1160 1165

Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg

1170 1175 1180

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

1185 1190 1195 1200

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

1205 1210 1215

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

1220 1225 1230

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

1235 1240 1245

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

1250 1255 1260

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

1265 1270 1275 1280

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

1285 1290 1295

Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser

1300 1305 1310

Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser

1315 1320 1325

Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly

1330 1335 1340

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

1345 1350 1355 1360

Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys

1365 1370 1375

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

1380 1385 1390

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

1395 1400 1405

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

1410 1415 1420

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

1425 1430 1435 1440

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

1445 1450 1455

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

1460 1465 1470

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1475 1480 1485

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1490 1495 1500

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1505 1510 1515 1520

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1525 1530 1535

Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg

1540 1545 1550

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1555 1560 1565

Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr

1570 1575 1580

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1585 1590 1595 1600

Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu

1605 1610 1615

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1620 1625 1630

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1635 1640 1645

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1650 1655 1660

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1665 1670 1675 1680

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1685 1690 1695

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1700 1705 1710

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1715 1720 1725

Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1730 1735 1740

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1745 1750 1755 1760

Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1765 1770 1775

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1780 1785 1790

Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1795 1800 1805

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1810 1815 1820

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1825 1830 1835 1840

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1845 1850 1855

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1860 1865 1870

Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1875 1880 1885

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1890 1895 1900

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1905 1910 1915 1920

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1925 1930 1935

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys

1940 1945 1950

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu

1955 1960 1965

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1970 1975 1980

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Gly

1985 1990 1995 2000

Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr

2005 2010 2015

Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu

2020 2025 2030

Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His

2035 2040 2045

Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser

2050 2055 2060

Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn

2065 2070 2075 2080

Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly Ser Gly

2085 2090 2095

Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln

2100 2105 2110

Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu

2115 2120 2125

Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr

2130 2135 2140

Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro

2145 2150 2155 2160

Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn

2165 2170 2175

Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser

2180 2185 2190

Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

2195 2200

<210> 2

<211> 6609

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 2

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctcc 60

tcagagactg ggcctgtcgc cgtcgatcca accctgcgcc gccggattga acctcacgag 120

tttgaagtgt tctttgaccc ccgggagctg agaaaggaga catgcctgct gtacgagatc 180

aactggggag gcaggcactc catctggagg cacacctctc agaacacaaa taagcacgtg 240

gaggtgaact tcatcgagaa gtttaccaca gagcggtact tctgccccaa taccagatgt 300

agcatcacat ggtttctgag ctggtcccct tgcggagagt gtagcagggc catcaccgag 360

ttcctgtcca gatatccaca cgtgacactg tttatctaca tcgccaggct gtatcaccac 420

gcagacccaa ggaataggca gggcctgcgc gatctgatca gctccggcgt gaccatccag 480

atcatgacag agcaggagtc cggctactgc tggcggaact tcgtgaatta ttctcctagc 540

aacgaggccc actggcctag gtacccacac ctgtgggtgc gcctgtacgt gctggagctg 600

tattgcatca tcctgggcct gcccccttgt ctgaatatcc tgcggagaaa gcagccccag 660

ctgaccttct ttacaatcgc cctgcagtct tgtcactatc agaggctgcc accccacatc 720

ctgtgggcca caggcctgaa gtctggagga tctagcggag gatcctctgg cagcgagaca 780

ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagcatg 840

tacaacatga tggagacgga gctgaagccg ccgggcccgc agcaaacttc ggggggcggc 900

ggcggcaact ccaccgcggc ggcggccggc ggcaaccaga aaaacagccc ggaccgcgtc 960

aagcggccca tgaatgcctt catggtgtgg tcccgcgggc agcggcgcaa gatggcccag 1020

gagaacccca agatgcacaa ctcggagatc agcaagcgcc tgggcgccga gtggaaactt 1080

ttgtcggaga cggagaagcg gccgttcatc gacgaggcta agcggctgcg agcgctgcac 1140

atgaaggagc acccggatta taaataccgg ccccggcgga aaaccaagac gctcatgaag 1200

aaggataagt acacgctgcc cggcgggctg ctggcccccg gcggcaatag catggcgagc 1260

ggggtcgggg tgggcgccgg cctgggcgcg ggcgtgaacc agcgcatgga cagttacgcg 1320

cacatgaacg gctggagcaa cggcagctac agcatgatgc aggaccagct gggctacccg 1380

cagcacccgg gcctcaatgc gcacggcgca gcgcagatgc agcccatgca ccgctacgac 1440

gtgagcgccc tgcagtacaa ctccatgacc agctcgcaga cctacatgaa cggctcgccc 1500

acctacagca tgtcctactc gcagcagggc acccctggca tggctcttgg ctccatgggt 1560

tcggtggtca agtccgaggc cagctccagc ccccctgtgg ttacctcttc ctcccactcc 1620

agggcgccct gccaggccgg ggacctccgg gacatgatca gcatgtatct ccccggcgcc 1680

gaggtgccgg aacccgccgc ccccagcaga cttcacatgt cccagcacta ccagagcggc 1740

ccggtgcccg gcacggccat taacggcaca ctgcccctct cacacatgag cggaggatct 1800

agcggaggat caagcggaag cgagactcct ggaaccagcg aaagcgcaac cccagaaagc 1860

agcggaggaa gtagcggagg aagcgacaag aagtacagca tcggcctggc catcggcacc 1920

aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 1980

gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc 2040

gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc 2100

agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 2160

gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac 2220

gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 2280

accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg 2340

atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac 2400

ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 2460

cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct 2520

gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag 2580

aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 2640

agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 2700

gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc 2760

aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 2820

aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 2880

ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac 2940

cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac 3000

aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 3060

aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag 3120

atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg 3180

aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc 3240

cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 3300

accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag 3360

cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 3420

ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga 3480

atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc 3540

aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag 3600

tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca 3660

taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag 3720

gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag 3780

gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg 3840

cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag 3900

cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc 3960

atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg 4020

tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt 4080

aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg 4140

cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga 4200

cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc 4260

cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg 4320

tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg 4380

tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac 4440

aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa 4500

gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc 4560

cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag 4620

gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag 4680

atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg 4740

aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac 4800

aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg 4860

ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac 4920

aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc 4980

gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc 5040

aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg 5100

tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat 5160

atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag 5220

aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc 5280

ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag 5340

tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc 5400

ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac 5460

ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg 5520

ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg 5580

aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag 5640

cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc 5700

agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc 5760

tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt 5820

accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg 5880

aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc 5940

ggcctgtacg agacacggat cgacctgtct cagctgggag gtgacagcgg cgggagcggc 6000

gggagcgggg ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg 6060

gtcattcagg agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag 6120

ccagagtctg acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg 6180

ctgctgacct ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac 6240

ggcgagaata agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac 6300

ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg 6360

atgctgcccg aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc 6420

cataccgcct acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca 6480

gagtataagc cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg 6540

ctgtctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg 6600

aaagtctaa 6609

<210> 3

<211> 2224

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 3

Met Pro Lys Lys Lys Arg Lys Val Met Tyr Asn Met Met Glu Thr Glu

1 5 10 15

Leu Lys Pro Pro Gly Pro Gln Gln Thr Ser Gly Gly Gly Gly Gly Asn

20 25 30

Ser Thr Ala Ala Ala Ala Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg

35 40 45

Val Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser Arg Gly Gln Arg

50 55 60

Arg Lys Met Ala Gln Glu Asn Pro Lys Met His Asn Ser Glu Ile Ser

65 70 75 80

Lys Arg Leu Gly Ala Glu Trp Lys Leu Leu Ser Glu Thr Glu Lys Arg

85 90 95

Pro Phe Ile Asp Glu Ala Lys Arg Leu Arg Ala Leu His Met Lys Glu

100 105 110

His Pro Asp Tyr Lys Tyr Arg Pro Arg Arg Lys Thr Lys Thr Leu Met

115 120 125

Lys Lys Asp Lys Tyr Thr Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly

130 135 140

Asn Ser Met Ala Ser Gly Val Gly Val Gly Ala Gly Leu Gly Ala Gly

145 150 155 160

Val Asn Gln Arg Met Asp Ser Tyr Ala His Met Asn Gly Trp Ser Asn

165 170 175

Gly Ser Tyr Ser Met Met Gln Asp Gln Leu Gly Tyr Pro Gln His Pro

180 185 190

Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln Pro Met His Arg Tyr

195 200 205

Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr

210 215 220

Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr

225 230 235 240

Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val Val Lys Ser Glu Ala

245 250 255

Ser Ser Ser Pro Pro Val Val Thr Ser Ser Ser His Ser Arg Ala Pro

260 265 270

Cys Gln Ala Gly Asp Leu Arg Asp Met Ile Ser Met Tyr Leu Pro Gly

275 280 285

Ala Glu Val Pro Glu Pro Ala Ala Pro Ser Arg Leu His Met Ser Gln

290 295 300

His Tyr Gln Ser Gly Pro Val Pro Gly Thr Ala Ile Asn Gly Thr Leu

305 310 315 320

Pro Leu Ser His Met Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser

325 330 335

Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly

340 345 350

Ser Ser Gly Gly Ser Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro

355 360 365

Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp

370 375 380

Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp

385 390 395 400

Gly Gly Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys

405 410 415

His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe

420 425 430

Cys Pro Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro

435 440 445

Cys Gly Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro

450 455 460

His Val Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp

465 470 475 480

Pro Arg Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr

485 490 495

Ile Gln Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe

500 505 510

Val Asn Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His

515 520 525

Leu Trp Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly

530 535 540

Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr

545 550 555 560

Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro

565 570 575

His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Ser Glu Thr Pro Gly

580 585 590

Thr Ser Glu Ser Ala Thr Pro Glu Leu Lys Asp Lys Lys Tyr Ser Ile

595 600 605

Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp

610 615 620

Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp

625 630 635 640

Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser

645 650 655

Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg

660 665 670

Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser

675 680 685

Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu

690 695 700

Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe

705 710 715 720

Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile

725 730 735

Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu

740 745 750

Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His

755 760 765

Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys

770 775 780

Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn

785 790 795 800

Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg

805 810 815

Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly

820 825 830

Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly

835 840 845

Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys

850 855 860

Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu

865 870 875 880

Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn

885 890 895

Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu

900 905 910

Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu

915 920 925

His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu

930 935 940

Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr

945 950 955 960

Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe

965 970 975

Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val

980 985 990

Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn

995 1000 1005

Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu

1010 1015 1020

Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys

1025 1030 1035 1040

Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu

1045 1050 1055

Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu

1060 1065 1070

Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser

1075 1080 1085

Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro

1090 1095 1100

Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr

1105 1110 1115 1120

Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg

1125 1130 1135

Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu

1140 1145 1150

Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp

1155 1160 1165

Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val

1170 1175 1180

Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys

1185 1190 1195 1200

Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile

1205 1210 1215

Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met

1220 1225 1230

Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val

1235 1240 1245

Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser

1250 1255 1260

Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile

1265 1270 1275 1280

Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln

1285 1290 1295

Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala

1300 1305 1310

Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu

1315 1320 1325

Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val

1330 1335 1340

Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile

1345 1350 1355 1360

Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys

1365 1370 1375

Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu

1380 1385 1390

Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln

1395 1400 1405

Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr

1410 1415 1420

Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp

1425 1430 1435 1440

His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys

1445 1450 1455

Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro

1460 1465 1470

Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu

1475 1480 1485

Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala

1490 1495 1500

Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg

1505 1510 1515 1520

Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu

1525 1530 1535

Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg

1540 1545 1550

Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg

1555 1560 1565

Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His

1570 1575 1580

Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys

1585 1590 1595 1600

Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val

1605 1610 1615

Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys

1620 1625 1630

Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys

1635 1640 1645

Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile

1650 1655 1660

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp

1665 1670 1675 1680

Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val

1685 1690 1695

Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu

1700 1705 1710

Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1715 1720 1725

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1730 1735 1740

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser

1745 1750 1755 1760

Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu

1765 1770 1775

Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys

1780 1785 1790

Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu

1795 1800 1805

Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly

1810 1815 1820

Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala

1825 1830 1835 1840

Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys

1845 1850 1855

Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu

1860 1865 1870

Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu

1875 1880 1885

Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg

1890 1895 1900

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly

1905 1910 1915 1920

Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg

1925 1930 1935

Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser

1940 1945 1950

Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1955 1960 1965

Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys

1970 1975 1980

Lys Thr Arg Asp Ser Gly Gly Ser Met Phe Gly Glu Ser Trp Lys Lys

1985 1990 1995 2000

His Leu Ser Gly Glu Phe Gly Lys Pro Tyr Phe Ile Lys Leu Met Gly

2005 2010 2015

Phe Val Ala Glu Glu Arg Lys His Tyr Thr Val Tyr Pro Pro Pro His

2020 2025 2030

Gln Val Phe Thr Trp Thr Gln Met Cys Asp Ile Lys Asp Val Lys Val

2035 2040 2045

Val Ile Leu Gly Gln Asp Pro Tyr His Gly Pro Asn Gln Ala His Gly

2050 2055 2060

Leu Cys Phe Ser Val Gln Arg Pro Val Pro Pro Pro Pro Ser Leu Glu

2065 2070 2075 2080

Asn Ile Tyr Lys Glu Leu Ser Thr Asp Ile Glu Asp Phe Val His Pro

2085 2090 2095

Gly His Gly Asp Leu Ser Gly Trp Ala Lys Gln Gly Val Leu Leu Leu

2100 2105 2110

Asn Ala Val Leu Thr Val Arg Ala His Gln Ala Asn Ser His Lys Glu

2115 2120 2125

Arg Gly Trp Glu Gln Phe Thr Asp Ala Val Val Ser Trp Leu Asn Gln

2130 2135 2140

Asn Ser Asn Gly Leu Val Phe Leu Leu Trp Gly Ser Tyr Ala Gln Lys

2145 2150 2155 2160

Lys Gly Ser Ala Ile Asp Arg Lys Arg His His Val Leu Gln Thr Ala

2165 2170 2175

His Pro Ser Pro Leu Ser Val Tyr Arg Gly Phe Phe Gly Cys Arg His

2180 2185 2190

Phe Ser Lys Thr Asn Glu Leu Leu Gln Lys Ser Gly Lys Lys Pro Ile

2195 2200 2205

Asp Trp Lys Glu Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

2210 2215 2220

<210> 4

<211> 6675

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 4

atgccaaaga agaagaggaa ggttatgtac aacatgatgg agacggagct gaagccgccg 60

ggcccgcagc aaacttcggg gggcggcggc ggcaactcca ccgcggcggc ggccggcggc 120

aaccagaaaa acagcccgga ccgcgtcaag cggcccatga atgccttcat ggtgtggtcc 180

cgcgggcagc ggcgcaagat ggcccaggag aaccccaaga tgcacaactc ggagatcagc 240

aagcgcctgg gcgccgagtg gaaacttttg tcggagacgg agaagcggcc gttcatcgac 300

gaggctaagc ggctgcgagc gctgcacatg aaggagcacc cggattataa ataccggccc 360

cggcggaaaa ccaagacgct catgaagaag gataagtaca cgctgcccgg cgggctgctg 420

gcccccggcg gcaatagcat ggcgagcggg gtcggggtgg gcgccggcct gggcgcgggc 480

gtgaaccagc gcatggacag ttacgcgcac atgaacggct ggagcaacgg cagctacagc 540

atgatgcagg accagctggg ctacccgcag cacccgggcc tcaatgcgca cggcgcagcg 600

cagatgcagc ccatgcaccg ctacgacgtg agcgccctgc agtacaactc catgaccagc 660

tcgcagacct acatgaacgg ctcgcccacc tacagcatgt cctactcgca gcagggcacc 720

cctggcatgg ctcttggctc catgggttcg gtggtcaagt ccgaggccag ctccagcccc 780

cctgtggtta cctcttcctc ccactccagg gcgccctgcc aggccgggga cctccgggac 840

atgatcagca tgtatctccc cggcgccgag gtgccggaac ccgccgcccc cagcagactt 900

cacatgtccc agcactacca gagcggcccg gtgcccggca cggccattaa cggcacactg 960

cccctctcac acatgagcgg aggatctagc ggaggatcaa gcggaagcga gactcctgga 1020

accagcgaaa gcgcaacccc agaaagcagc ggaggaagta gcggaggaag ctcatcggag 1080

accggccctg ttgctgttga ccccaccctg cggcggagaa tcgagccaca cgagttcgag 1140

gtgttcttcg acccaaggga gctccgcaag gagacgtgcc tcctgtacga gatcaactgg 1200

ggcggcaggc actccatctg gaggcacacc agccaaaaca ccaacaagca cgtggaggtc 1260

aacttcatcg agaagttcac caccgagagg tacttctgcc caaacacccg ctgctccatc 1320

acctggttcc tgtcctggag cccatgcggc gagtgctcca gggccatcac cgagttcctc 1380

agccgctacc cacacgtcac cctgttcatc tacatcgcca ggctctacca ccacgccgac 1440

ccaaggaaca ggcagggcct ccgcgacctg atctccagcg gcgtgaccat ccaaatcatg 1500

accgagcagg agtccggcta ctgctggagg aacttcgtca actactcccc aagcaacgag 1560

gcccactggc caaggtaccc acacctctgg gtgcgcctct acgtgctcga gctgtactgc 1620

atcatcctcg gcctgccacc atgcctcaac atcctgaggc gcaagcaacc acagctgacc 1680

ttcttcacca tcgccctcca aagctgccac taccagaggc tcccaccaca catcctgtgg 1740

gctaccggcc tcaagtccgg cagcgagacg ccaggcacct ccgagagcgc tacgcctgaa 1800

cttaaggaca agaagtactc gatcggcctc gccatcggga cgaactcagt tggctgggcc 1860

gtgatcaccg acgagtacaa ggtgccctct aagaagttca aggtcctggg gaacaccgac 1920

cgccattcca tcaagaagaa cctcatcggc gctctcctgt tcgacagcgg ggagaccgct 1980

gaggctacga ggctcaagag aaccgctagg cgccggtaca cgagaaggaa gaacaggatc 2040

tgctacctcc aagagatttt ctccaacgag atggccaagg ttgacgattc attcttccac 2100

cgcctggagg agtctttcct cgtggaggag gataagaagc acgagcggca tcccatcttc 2160

ggcaacatcg tggacgaggt tgcctaccac gagaagtacc ctacgatcta ccatctgcgg 2220

aagaagctcg tggactccac cgataaggcg gacctcagac tgatctacct cgctctggcc 2280

cacatgatca agttccgcgg ccatttcctg atcgaggggg atctcaaccc agacaacagc 2340

gatgttgaca agctgttcat ccaactcgtg cagacctaca accaactctt cgaggagaac 2400

ccgatcaacg cctctggcgt ggacgcgaag gctatcctgt ccgcgaggct ctcgaagtcc 2460

aggaggctgg agaacctgat cgctcagctc ccaggcgaga agaagaacgg cctgttcggg 2520

aacctcatcg ctctcagcct ggggctcacc ccgaacttca agtcgaactt cgatctcgct 2580

gaggacgcca agctgcaact ctccaaggac acctacgacg atgacctcga taacctcctg 2640

gcccagatcg gcgatcaata cgcggacctg ttcctcgctg ccaagaacct gtcggacgcc 2700

atcctcctgt cagatatcct ccgcgtgaac accgagatca cgaaggctcc actctctgcc 2760

tccatgatca agcgctacga cgagcaccat caggatctga ccctcctgaa ggcgctggtc 2820

cgccaacagc tcccggagaa gtacaaggag attttcttcg atcagtcgaa gaacggctac 2880

gctgggtaca tcgacggcgg ggcctcacaa gaggagttct acaagttcat caagccaatc 2940

ctggagaaga tggacggcac ggaggagctc ctggtgaagc tcaacaggga ggacctcctg 3000

cggaagcaga gaaccttcga taacggcagc atcccccacc aaatccatct cggggagctg 3060

cacgccatcc tgagaaggca agaggacttc taccctttcc tcaaggataa ccgggagaag 3120

atcgagaaga tcctgacctt cagaatccca tactacgtcg gccctctcgc gcgggggaac 3180

tcaagattcg cttggatgac ccgcaagtct gaggagacca tcacgccgtg gaacttcgag 3240

gaggtggtgg acaagggcgc tagcgctcag tcgttcatcg agaggatgac caacttcgac 3300

aagaacctgc ccaacgagaa ggtgctccct aagcactcgc tcctgtacga gtacttcacc 3360

gtctacaacg agctcacgaa ggtgaagtac gtcaccgagg gcatgcgcaa gccagcgttc 3420

ctgtccgggg agcagaagaa ggctatcgtg gacctcctgt tcaagaccaa ccggaaggtc 3480

acggttaagc aactcaagga ggactacttc aagaagatcg agtgcttcga ttcggtcgag 3540

atcagcggcg ttgaggaccg cttcaacgcc agcctcggga cctaccacga tctcctgaag 3600

atcatcaagg ataaggactt cctggacaac gaggagaacg aggatatcct ggaggacatc 3660

gtgctgaccc tcacgctgtt cgaggacagg gagatgatcg aggagcgcct gaagacgtac 3720

gcccatctct tcgatgacaa ggtcatgaag caactcaagc gccggagata caccggctgg 3780

gggaggctgt cccgcaagct catcaacggc atccgggaca agcagtccgg gaagaccatc 3840

ctcgacttcc tcaagagcga tggcttcgcc aacaggaact tcatgcaact gatccacgat 3900

gacagcctca ccttcaagga ggatatccaa aaggctcaag tgagcggcca gggggactcg 3960

ctgcacgagc atatcgcgaa cctcgctggc tcccccgcga tcaagaaggg catcctccag 4020

accgtgaagg ttgtggacga gctcgtgaag gtcatgggcc ggcacaagcc tgagaacatc 4080

gtcatcgaga tggccagaga gaaccaaacc acgcagaagg ggcaaaagaa ctctagggag 4140

cgcatgaagc gcatcgagga gggcatcaag gagctggggt cccaaatcct caaggagcac 4200

ccagtggaga acacccaact gcagaacgag aagctctacc tgtactacct ccagaacggc 4260

agggatatgt acgtggacca agagctggat atcaaccgcc tcagcgatta cgacgtcgat 4320

catatcgttc cccagtcttt cctgaaggat gactccatcg acaacaaggt cctcaccagg 4380

tcggacaaga accgcggcaa gtcagataac gttccatctg aggaggtcgt taagaagatg 4440

aagaactact ggaggcagct cctgaacgcc aagctgatca cgcaaaggaa gttcgacaac 4500

ctcaccaagg ctgagagagg cgggctctca gagctggaca aggccggctt catcaagcgg 4560

cagctggtcg agaccagaca aatcacgaag cacgttgcgc aaatcctcga ctctcggatg 4620

aacacgaagt acgatgagaa cgacaagctg atcagggagg ttaaggtgat caccctgaag 4680

tctaagctcg tctccgactt caggaaggat ttccagttct acaaggttcg cgagatcaac 4740

aactaccacc atgcccatga cgcttacctc aacgctgtgg tcggcaccgc tctgatcaag 4800

aagtacccaa agctggagtc cgagttcgtg tacggggact acaaggttta cgatgtgcgc 4860

aagatgatcg ccaagtcgga gcaagagatc ggcaaggcta ccgccaagta cttcttctac 4920

tcaaacatca tgaacttctt caagaccgag atcacgctgg ccaacggcga gatccggaag 4980

agaccgctca tcgagaccaa cggcgagacg ggggagatcg tgtgggacaa gggcagggat 5040

ttcgcgaccg tccgcaaggt tctctccatg ccccaggtga acatcgtcaa gaagaccgag 5100

gtccaaacgg gcgggttctc aaaggagtct atcctgccta agcggaacag cgacaagctc 5160

atcgccagaa agaaggactg ggacccaaag aagtacggcg ggttcgacag ccctaccgtg 5220

gcctactcgg tcctggttgt ggcgaaggtt gagaagggca agtccaagaa gctcaagagc 5280

gtgaaggagc tcctggggat caccatcatg gagaggtcca gcttcgagaa gaacccaatc 5340

gacttcctgg aggccaaggg ctacaaggag gtgaagaagg acctgatcat caagctcccg 5400

aagtactctc tcttcgagct ggagaacggc aggaagagaa tgctggcttc cgctggcgag 5460

ctccagaagg ggaacgagct cgcgctgcca agcaagtacg tgaacttcct ctacctggct 5520

tcccactacg agaagctcaa gggcagcccg gaggacaacg agcaaaagca gctgttcgtc 5580

gagcagcaca agcattacct cgacgagatc atcgagcaaa tctccgagtt cagcaagcgc 5640

gtgatcctcg ccgacgcgaa cctggataag gtcctctccg cctacaacaa gcaccgggac 5700

aagcccatca gagagcaagc ggagaacatc atccatctct tcaccctgac gaacctcggc 5760

gctcctgctg ctttcaagta cttcgacacc acgatcgatc ggaagagata cacctccacg 5820

aaggaggtcc tggacgcgac cctcatccac cagtcgatca ccggcctgta cgagacgagg 5880

atcgacctct cacaactcgg cggggataag agacccgcag caaccaagaa ggcagggcaa 5940

gcaaagaaga agaagacgcg tgactccggc ggcagcatgt ttggagagag ctggaagaag 6000

cacctcagcg gggagttcgg gaaaccgtat tttatcaagc taatgggatt tgttgcagaa 6060

gaaagaaagc attacactgt ttatccaccc ccacaccaag tcttcacctg gacccagatg 6120

tgtgacataa aagatgtgaa ggttgtcatc ctgggacagg atccatatca tggacctaat 6180

caagctcacg ggctctgctt tagtgttcaa aggcctgttc cgcctccgcc cagtttggag 6240

aacatttata aagagttgtc tacagacata gaggattttg ttcatcctgg ccatggagat 6300

ttatctgggt gggccaagca aggtgttctc cttctcaacg ctgtcctcac ggttcgtgcc 6360

catcaagcca actctcataa ggagcgaggc tgggagcagt tcactgatgc agttgtgtcc 6420

tggctaaatc agaactcgaa tggccttgtt ttcttgctct ggggctctta tgctcagaag 6480

aagggcagtg ccattgatag gaagcggcac catgtactac agacggctca tccctcccct 6540

ttgtcagtgt atagagggtt ctttggatgt agacactttt caaagaccaa tgagctgctg 6600

cagaagtctg gcaagaagcc cattgactgg aaggagctgt cgggggggag cccaaagaag 6660

aagcggaagg tgtag 6675

<210> 5

<211> 204

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

ccctcctacg ttgcggtcac acccttctcc cttcggggag acaacgacgg cggtggcggg 60

agcttctcca cggccgacca gctggagatg gtgaccgagc tgctgggagg agacatggtg 120

aaccagagtt tcatctgcga cccggacgac gagaccttca tcaaaaacat catcatccag 180

gactgtatgt ggagcggctt ctcg 204

<210> 6

<211> 5871

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctcc 60

tcagagactg ggcctgtcgc cgtcgatcca accctgcgcc gccggattga acctcacgag 120

tttgaagtgt tctttgaccc ccgggagctg agaaaggaga catgcctgct gtacgagatc 180

aactggggag gcaggcactc catctggagg cacacctctc agaacacaaa taagcacgtg 240

gaggtgaact tcatcgagaa gtttaccaca gagcggtact tctgccccaa taccagatgt 300

agcatcacat ggtttctgag ctggtcccct tgcggagagt gtagcagggc catcaccgag 360

ttcctgtcca gatatccaca cgtgacactg tttatctaca tcgccaggct gtatcaccac 420

gcagacccaa ggaataggca gggcctgcgc gatctgatca gctccggcgt gaccatccag 480

atcatgacag agcaggagtc cggctactgc tggcggaact tcgtgaatta ttctcctagc 540

aacgaggccc actggcctag gtacccacac ctgtgggtgc gcctgtacgt gctggagctg 600

tattgcatca tcctgggcct gcccccttgt ctgaatatcc tgcggagaaa gcagccccag 660

ctgaccttct ttacaatcgc cctgcagtct tgtcactatc agaggctgcc accccacatc 720

ctgtgggcca caggcctgaa gtctggagga tctagcggag gatcctctgg cagcgagaca 780

ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagccgc 840

gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc 900

caggagaacc ccaagatgca caactcggag atcagcaagc gcctgggcgc cgagtggaaa 960

cttttgtcgg agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg 1020

cacatgaagg agcacccgga ttataaatac cggagcggag gatctagcgg aggatcaagc 1080

ggaagcgaga ctcctggaac cagcgaaagc gcaaccccag aaagcagcgg aggaagtagc 1140

ggaggaagcg acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg 1200

gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc 1260

gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca 1320

gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg 1380

atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc 1440

cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc 1500

ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg 1560

agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg 1620

gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac 1680

agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa 1740

aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag 1800

agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc 1860

ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg 1920

gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg 1980

ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac 2040

gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc 2100

gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc 2160

gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc 2220

tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc 2280

atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg 2340

ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag 2400

ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa 2460

aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga 2520

aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc 2580

gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc 2640

gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc 2700

accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc 2760

ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa 2820

gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg 2880

gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg 2940

aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat 3000

atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc 3060

tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc 3120

tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca 3180

atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac 3240

gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat 3300

agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg 3360

cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac 3420

atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc 3480

gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa 3540

caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat 3600

gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg 3660

gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc 3720

agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag 3780

atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac 3840

aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag 3900

agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg 3960

atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg 4020

aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc 4080

aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc 4140

aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg 4200

cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc 4260

tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg 4320

aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg 4380

gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc 4440

gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag 4500

ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc 4560

gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag 4620

agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc 4680

atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg 4740

cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc 4800

gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg 4860

gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt 4920

gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag 4980

agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg 5040

gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg 5100

ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc 5160

accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca 5220

cggatcgacc tgtctcagct gggaggtgac agcggcggga gcggcgggag cggggggagc 5280

actaatctga gcgacatcat tgagaaggag actgggaaac agctggtcat tcaggagtcc 5340

atcctgatgc tgcctgagga ggtggaggaa gtgatcggca acaagccaga gtctgacatc 5400

ctggtgcaca ccgcctacga cgagtccaca gatgagaatg tgatgctgct gacctctgac 5460

gcccccgagt ataagccttg ggccctggtc atccaggatt ctaacggcga gaataagatc 5520

aagatgctga gcggaggatc cggaggatct ggaggcagca ccaacctgtc tgacatcatc 5580

gagaaggaga caggcaagca gctggtcatc caggagagca tcctgatgct gcccgaagaa 5640

gtcgaagaag tgatcggaaa caagcctgag agcgatatcc tggtccatac cgcctacgac 5700

gagagtaccg acgaaaatgt gatgctgctg acatccgacg ccccagagta taagccctgg 5760

gctctggtca tccaggattc caacggagag aacaaaatca aaatgctgtc tggcggctca 5820

aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt c 5871

<210> 7

<211> 6006

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 7

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctcc 60

tcagagactg ggcctgtcgc cgtcgatcca accctgcgcc gccggattga acctcacgag 120

tttgaagtgt tctttgaccc ccgggagctg agaaaggaga catgcctgct gtacgagatc 180

aactggggag gcaggcactc catctggagg cacacctctc agaacacaaa taagcacgtg 240

gaggtgaact tcatcgagaa gtttaccaca gagcggtact tctgccccaa taccagatgt 300

agcatcacat ggtttctgag ctggtcccct tgcggagagt gtagcagggc catcaccgag 360

ttcctgtcca gatatccaca cgtgacactg tttatctaca tcgccaggct gtatcaccac 420

gcagacccaa ggaataggca gggcctgcgc gatctgatca gctccggcgt gaccatccag 480

atcatgacag agcaggagtc cggctactgc tggcggaact tcgtgaatta ttctcctagc 540

aacgaggccc actggcctag gtacccacac ctgtgggtgc gcctgtacgt gctggagctg 600

tattgcatca tcctgggcct gcccccttgt ctgaatatcc tgcggagaaa gcagccccag 660

ctgaccttct ttacaatcgc cctgcagtct tgtcactatc agaggctgcc accccacatc 720

ctgtgggcca caggcctgaa gtctggagga tctagcggag gatcctctgg cagcgagaca 780

ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagcgac 840

gtgagcgccc tgcagtacaa ctccatgacc agctcgcaga cctacatgaa cggctcgccc 900

acctacagca tgtcctactc gcagcagggc acccctggca tggctcttgg ctccatgggt 960

tcggtggtca agtccgaggc cagctccagc ccccctgtgg ttacctcttc ctcccactcc 1020

agggcgccct gccaggccgg ggacctccgg gacatgatca gcatgtatct ccccggcgcc 1080

gaggtgccgg aacccgccgc ccccagcaga cttcacatgt cccagcacta ccagagcggc 1140

ccggtgcccg gcacggccat taacggcaca ctgcccctct cacacatgag cggaggatct 1200

agcggaggat caagcggaag cgagactcct ggaaccagcg aaagcgcaac cccagaaagc 1260

agcggaggaa gtagcggagg aagcgacaag aagtacagca tcggcctggc catcggcacc 1320

aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 1380

gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc 1440

gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc 1500

agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 1560

gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac 1620

gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 1680

accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg 1740

atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac 1800

ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 1860

cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct 1920

gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag 1980

aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 2040

agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 2100

gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc 2160

aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 2220

aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 2280

ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac 2340

cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac 2400

aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 2460

aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag 2520

atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg 2580

aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc 2640

cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 2700

accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag 2760

cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 2820

ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga 2880

atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc 2940

aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag 3000

tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca 3060

taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag 3120

gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag 3180

gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg 3240

cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag 3300

cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc 3360

atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg 3420

tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt 3480

aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg 3540

cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga 3600

cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc 3660

cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg 3720

tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg 3780

tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac 3840

aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa 3900

gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc 3960

cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag 4020

gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag 4080

atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg 4140

aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac 4200

aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg 4260

ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac 4320

aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc 4380

gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc 4440

aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg 4500

tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat 4560

atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag 4620

aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc 4680

ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag 4740

tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc 4800

ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac 4860

ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg 4920

ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg 4980

aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag 5040

cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc 5100

agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc 5160

tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt 5220

accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg 5280

aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc 5340

ggcctgtacg agacacggat cgacctgtct cagctgggag gtgacagcgg cgggagcggc 5400

gggagcgggg ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg 5460

gtcattcagg agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag 5520

ccagagtctg acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg 5580

ctgctgacct ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac 5640

ggcgagaata agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac 5700

ctgtctgaca tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg 5760

atgctgcccg aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc 5820

cataccgcct acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca 5880

gagtataagc cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg 5940

ctgtctggcg gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg 6000

aaagtc 6006

<210> 8

<211> 6072

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 8

atgccaaaga agaagaggaa ggttgacgtg agcgccctgc agtacaactc catgaccagc 60

tcgcagacct acatgaacgg ctcgcccacc tacagcatgt cctactcgca gcagggcacc 120

cctggcatgg ctcttggctc catgggttcg gtggtcaagt ccgaggccag ctccagcccc 180

cctgtggtta cctcttcctc ccactccagg gcgccctgcc aggccgggga cctccgggac 240

atgatcagca tgtatctccc cggcgccgag gtgccggaac ccgccgcccc cagcagactt 300

cacatgtccc agcactacca gagcggcccg gtgcccggca cggccattaa cggcacactg 360

cccctctcac acatgagcgg aggatctagc ggaggatcaa gcggaagcga gactcctgga 420

accagcgaaa gcgcaacccc agaaagcagc ggaggaagta gcggaggaag ctcatcggag 480

accggccctg ttgctgttga ccccaccctg cggcggagaa tcgagccaca cgagttcgag 540

gtgttcttcg acccaaggga gctccgcaag gagacgtgcc tcctgtacga gatcaactgg 600

ggcggcaggc actccatctg gaggcacacc agccaaaaca ccaacaagca cgtggaggtc 660

aacttcatcg agaagttcac caccgagagg tacttctgcc caaacacccg ctgctccatc 720

acctggttcc tgtcctggag cccatgcggc gagtgctcca gggccatcac cgagttcctc 780

agccgctacc cacacgtcac cctgttcatc tacatcgcca ggctctacca ccacgccgac 840

ccaaggaaca ggcagggcct ccgcgacctg atctccagcg gcgtgaccat ccaaatcatg 900

accgagcagg agtccggcta ctgctggagg aacttcgtca actactcccc aagcaacgag 960

gcccactggc caaggtaccc acacctctgg gtgcgcctct acgtgctcga gctgtactgc 1020

atcatcctcg gcctgccacc atgcctcaac atcctgaggc gcaagcaacc acagctgacc 1080

ttcttcacca tcgccctcca aagctgccac taccagaggc tcccaccaca catcctgtgg 1140

gctaccggcc tcaagtccgg cagcgagacg ccaggcacct ccgagagcgc tacgcctgaa 1200

cttaaggaca agaagtactc gatcggcctc gccatcggga cgaactcagt tggctgggcc 1260

gtgatcaccg acgagtacaa ggtgccctct aagaagttca aggtcctggg gaacaccgac 1320

cgccattcca tcaagaagaa cctcatcggc gctctcctgt tcgacagcgg ggagaccgct 1380

gaggctacga ggctcaagag aaccgctagg cgccggtaca cgagaaggaa gaacaggatc 1440

tgctacctcc aagagatttt ctccaacgag atggccaagg ttgacgattc attcttccac 1500

cgcctggagg agtctttcct cgtggaggag gataagaagc acgagcggca tcccatcttc 1560

ggcaacatcg tggacgaggt tgcctaccac gagaagtacc ctacgatcta ccatctgcgg 1620

aagaagctcg tggactccac cgataaggcg gacctcagac tgatctacct cgctctggcc 1680

cacatgatca agttccgcgg ccatttcctg atcgaggggg atctcaaccc agacaacagc 1740

gatgttgaca agctgttcat ccaactcgtg cagacctaca accaactctt cgaggagaac 1800

ccgatcaacg cctctggcgt ggacgcgaag gctatcctgt ccgcgaggct ctcgaagtcc 1860

aggaggctgg agaacctgat cgctcagctc ccaggcgaga agaagaacgg cctgttcggg 1920

aacctcatcg ctctcagcct ggggctcacc ccgaacttca agtcgaactt cgatctcgct 1980

gaggacgcca agctgcaact ctccaaggac acctacgacg atgacctcga taacctcctg 2040

gcccagatcg gcgatcaata cgcggacctg ttcctcgctg ccaagaacct gtcggacgcc 2100

atcctcctgt cagatatcct ccgcgtgaac accgagatca cgaaggctcc actctctgcc 2160

tccatgatca agcgctacga cgagcaccat caggatctga ccctcctgaa ggcgctggtc 2220

cgccaacagc tcccggagaa gtacaaggag attttcttcg atcagtcgaa gaacggctac 2280

gctgggtaca tcgacggcgg ggcctcacaa gaggagttct acaagttcat caagccaatc 2340

ctggagaaga tggacggcac ggaggagctc ctggtgaagc tcaacaggga ggacctcctg 2400

cggaagcaga gaaccttcga taacggcagc atcccccacc aaatccatct cggggagctg 2460

cacgccatcc tgagaaggca agaggacttc taccctttcc tcaaggataa ccgggagaag 2520

atcgagaaga tcctgacctt cagaatccca tactacgtcg gccctctcgc gcgggggaac 2580

tcaagattcg cttggatgac ccgcaagtct gaggagacca tcacgccgtg gaacttcgag 2640

gaggtggtgg acaagggcgc tagcgctcag tcgttcatcg agaggatgac caacttcgac 2700

aagaacctgc ccaacgagaa ggtgctccct aagcactcgc tcctgtacga gtacttcacc 2760

gtctacaacg agctcacgaa ggtgaagtac gtcaccgagg gcatgcgcaa gccagcgttc 2820

ctgtccgggg agcagaagaa ggctatcgtg gacctcctgt tcaagaccaa ccggaaggtc 2880

acggttaagc aactcaagga ggactacttc aagaagatcg agtgcttcga ttcggtcgag 2940

atcagcggcg ttgaggaccg cttcaacgcc agcctcggga cctaccacga tctcctgaag 3000

atcatcaagg ataaggactt cctggacaac gaggagaacg aggatatcct ggaggacatc 3060

gtgctgaccc tcacgctgtt cgaggacagg gagatgatcg aggagcgcct gaagacgtac 3120

gcccatctct tcgatgacaa ggtcatgaag caactcaagc gccggagata caccggctgg 3180

gggaggctgt cccgcaagct catcaacggc atccgggaca agcagtccgg gaagaccatc 3240

ctcgacttcc tcaagagcga tggcttcgcc aacaggaact tcatgcaact gatccacgat 3300

gacagcctca ccttcaagga ggatatccaa aaggctcaag tgagcggcca gggggactcg 3360

ctgcacgagc atatcgcgaa cctcgctggc tcccccgcga tcaagaaggg catcctccag 3420

accgtgaagg ttgtggacga gctcgtgaag gtcatgggcc ggcacaagcc tgagaacatc 3480

gtcatcgaga tggccagaga gaaccaaacc acgcagaagg ggcaaaagaa ctctagggag 3540

cgcatgaagc gcatcgagga gggcatcaag gagctggggt cccaaatcct caaggagcac 3600

ccagtggaga acacccaact gcagaacgag aagctctacc tgtactacct ccagaacggc 3660

agggatatgt acgtggacca agagctggat atcaaccgcc tcagcgatta cgacgtcgat 3720

catatcgttc cccagtcttt cctgaaggat gactccatcg acaacaaggt cctcaccagg 3780

tcggacaaga accgcggcaa gtcagataac gttccatctg aggaggtcgt taagaagatg 3840

aagaactact ggaggcagct cctgaacgcc aagctgatca cgcaaaggaa gttcgacaac 3900

ctcaccaagg ctgagagagg cgggctctca gagctggaca aggccggctt catcaagcgg 3960

cagctggtcg agaccagaca aatcacgaag cacgttgcgc aaatcctcga ctctcggatg 4020

aacacgaagt acgatgagaa cgacaagctg atcagggagg ttaaggtgat caccctgaag 4080

tctaagctcg tctccgactt caggaaggat ttccagttct acaaggttcg cgagatcaac 4140

aactaccacc atgcccatga cgcttacctc aacgctgtgg tcggcaccgc tctgatcaag 4200

aagtacccaa agctggagtc cgagttcgtg tacggggact acaaggttta cgatgtgcgc 4260

aagatgatcg ccaagtcgga gcaagagatc ggcaaggcta ccgccaagta cttcttctac 4320

tcaaacatca tgaacttctt caagaccgag atcacgctgg ccaacggcga gatccggaag 4380

agaccgctca tcgagaccaa cggcgagacg ggggagatcg tgtgggacaa gggcagggat 4440

ttcgcgaccg tccgcaaggt tctctccatg ccccaggtga acatcgtcaa gaagaccgag 4500

gtccaaacgg gcgggttctc aaaggagtct atcctgccta agcggaacag cgacaagctc 4560

atcgccagaa agaaggactg ggacccaaag aagtacggcg ggttcgacag ccctaccgtg 4620

gcctactcgg tcctggttgt ggcgaaggtt gagaagggca agtccaagaa gctcaagagc 4680

gtgaaggagc tcctggggat caccatcatg gagaggtcca gcttcgagaa gaacccaatc 4740

gacttcctgg aggccaaggg ctacaaggag gtgaagaagg acctgatcat caagctcccg 4800

aagtactctc tcttcgagct ggagaacggc aggaagagaa tgctggcttc cgctggcgag 4860

ctccagaagg ggaacgagct cgcgctgcca agcaagtacg tgaacttcct ctacctggct 4920

tcccactacg agaagctcaa gggcagcccg gaggacaacg agcaaaagca gctgttcgtc 4980

gagcagcaca agcattacct cgacgagatc atcgagcaaa tctccgagtt cagcaagcgc 5040

gtgatcctcg ccgacgcgaa cctggataag gtcctctccg cctacaacaa gcaccgggac 5100

aagcccatca gagagcaagc ggagaacatc atccatctct tcaccctgac gaacctcggc 5160

gctcctgctg ctttcaagta cttcgacacc acgatcgatc ggaagagata cacctccacg 5220

aaggaggtcc tggacgcgac cctcatccac cagtcgatca ccggcctgta cgagacgagg 5280

atcgacctct cacaactcgg cggggataag agacccgcag caaccaagaa ggcagggcaa 5340

gcaaagaaga agaagacgcg tgactccggc ggcagcatgt ttggagagag ctggaagaag 5400

cacctcagcg gggagttcgg gaaaccgtat tttatcaagc taatgggatt tgttgcagaa 5460

gaaagaaagc attacactgt ttatccaccc ccacaccaag tcttcacctg gacccagatg 5520

tgtgacataa aagatgtgaa ggttgtcatc ctgggacagg atccatatca tggacctaat 5580

caagctcacg ggctctgctt tagtgttcaa aggcctgttc cgcctccgcc cagtttggag 5640

aacatttata aagagttgtc tacagacata gaggattttg ttcatcctgg ccatggagat 5700

ttatctgggt gggccaagca aggtgttctc cttctcaacg ctgtcctcac ggttcgtgcc 5760

catcaagcca actctcataa ggagcgaggc tgggagcagt tcactgatgc agttgtgtcc 5820

tggctaaatc agaactcgaa tggccttgtt ttcttgctct ggggctctta tgctcagaag 5880

aagggcagtg ccattgatag gaagcggcac catgtactac agacggctca tccctcccct 5940

ttgtcagtgt atagagggtt ctttggatgt agacactttt caaagaccaa tgagctgctg 6000

cagaagtctg gcaagaagcc cattgactgg aaggagctgt cgggggggag cccaaagaag 6060

aagcggaagg tg 6072

<210> 9

<211> 6612

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

atgccaaaga agaagaggaa ggttctggtt gggccctcct gtgtcatgga tgacttcagg 60

gacccacagc gatggaagga atgtgccaag caagggaaaa tgccatgtta ctttgatctt 120

attgaagaaa atgtttattt aacagaaaga aagaagaata aatctcatcg agatattaag 180

cgaatgcagt gtgagtgtac acctctttct aaagatgaaa gagctcaagg tgaaatagca 240

tgtggggaag attgtcttaa tcgtcttctc atgattgaat gttcttctcg gtgtccaaat 300

ggggattatt gttccaatag acggtttcag agaaaacagc atgcagatgt ggaagtcata 360

ctcacagaaa agaaaggctg gggcttgaga gctgccaaag accttccttc gaacaccttt 420

gtcctagaat attgtggaga ggtactcgat cataaagagt ttaaagctcg agtgaaggag 480

tatgcacgaa acaaaaacat ccattactat ttcatggccc tgaagaatga tgagataata 540

gatgccactc aaaaaggaaa ttgctctcgt ttcatgaatc acagctgtga accaaattgt 600

gaaacccaaa aatggactgt gaacggacaa ctgagggttg ggttttttac caccaaactg 660

gttccttcag gctcagagtt aacgtttgac tatcagttcc agagatatgg aaaagaagcc 720

cagaaatgtt tctgcggatc agccaattgc cggggttacc tgggaggaga aaacagagtc 780

agcatcagag cagcaggagg gaaaatgaag aaggaacgat ctcgtaagaa ggattcagtg 840

gatggagagc tagaagctct gatggaaaat ggtgagggtc tctctgataa aaaccaggtg 900

ctcagcttat cccggagcgg aggatctagc ggaggatcaa gcggaagcga gactcctgga 960

accagcgaaa gcgcaacccc agaaagcagc ggaggaagta gcggaggaag ctcatcggag 1020

accggccctg ttgctgttga ccccaccctg cggcggagaa tcgagccaca cgagttcgag 1080

gtgttcttcg acccaaggga gctccgcaag gagacgtgcc tcctgtacga gatcaactgg 1140

ggcggcaggc actccatctg gaggcacacc agccaaaaca ccaacaagca cgtggaggtc 1200

aacttcatcg agaagttcac caccgagagg tacttctgcc caaacacccg ctgctccatc 1260

acctggttcc tgtcctggag cccatgcggc gagtgctcca gggccatcac cgagttcctc 1320

agccgctacc cacacgtcac cctgttcatc tacatcgcca ggctctacca ccacgccgac 1380

ccaaggaaca ggcagggcct ccgcgacctg atctccagcg gcgtgaccat ccaaatcatg 1440

accgagcagg agtccggcta ctgctggagg aacttcgtca actactcccc aagcaacgag 1500

gcccactggc caaggtaccc acacctctgg gtgcgcctct acgtgctcga gctgtactgc 1560

atcatcctcg gcctgccacc atgcctcaac atcctgaggc gcaagcaacc acagctgacc 1620

ttcttcacca tcgccctcca aagctgccac taccagaggc tcccaccaca catcctgtgg 1680

gctaccggcc tcaagtccgg cagcgagacg ccaggcacct ccgagagcgc tacgcctgaa 1740

cttaaggaca agaagtactc gatcggcctc gccatcggga cgaactcagt tggctgggcc 1800

gtgatcaccg acgagtacaa ggtgccctct aagaagttca aggtcctggg gaacaccgac 1860

cgccattcca tcaagaagaa cctcatcggc gctctcctgt tcgacagcgg ggagaccgct 1920

gaggctacga ggctcaagag aaccgctagg cgccggtaca cgagaaggaa gaacaggatc 1980

tgctacctcc aagagatttt ctccaacgag atggccaagg ttgacgattc attcttccac 2040

cgcctggagg agtctttcct cgtggaggag gataagaagc acgagcggca tcccatcttc 2100

ggcaacatcg tggacgaggt tgcctaccac gagaagtacc ctacgatcta ccatctgcgg 2160

aagaagctcg tggactccac cgataaggcg gacctcagac tgatctacct cgctctggcc 2220

cacatgatca agttccgcgg ccatttcctg atcgaggggg atctcaaccc agacaacagc 2280

gatgttgaca agctgttcat ccaactcgtg cagacctaca accaactctt cgaggagaac 2340

ccgatcaacg cctctggcgt ggacgcgaag gctatcctgt ccgcgaggct ctcgaagtcc 2400

aggaggctgg agaacctgat cgctcagctc ccaggcgaga agaagaacgg cctgttcggg 2460

aacctcatcg ctctcagcct ggggctcacc ccgaacttca agtcgaactt cgatctcgct 2520

gaggacgcca agctgcaact ctccaaggac acctacgacg atgacctcga taacctcctg 2580

gcccagatcg gcgatcaata cgcggacctg ttcctcgctg ccaagaacct gtcggacgcc 2640

atcctcctgt cagatatcct ccgcgtgaac accgagatca cgaaggctcc actctctgcc 2700

tccatgatca agcgctacga cgagcaccat caggatctga ccctcctgaa ggcgctggtc 2760

cgccaacagc tcccggagaa gtacaaggag attttcttcg atcagtcgaa gaacggctac 2820

gctgggtaca tcgacggcgg ggcctcacaa gaggagttct acaagttcat caagccaatc 2880

ctggagaaga tggacggcac ggaggagctc ctggtgaagc tcaacaggga ggacctcctg 2940

cggaagcaga gaaccttcga taacggcagc atcccccacc aaatccatct cggggagctg 3000

cacgccatcc tgagaaggca agaggacttc taccctttcc tcaaggataa ccgggagaag 3060

atcgagaaga tcctgacctt cagaatccca tactacgtcg gccctctcgc gcgggggaac 3120

tcaagattcg cttggatgac ccgcaagtct gaggagacca tcacgccgtg gaacttcgag 3180

gaggtggtgg acaagggcgc tagcgctcag tcgttcatcg agaggatgac caacttcgac 3240

aagaacctgc ccaacgagaa ggtgctccct aagcactcgc tcctgtacga gtacttcacc 3300

gtctacaacg agctcacgaa ggtgaagtac gtcaccgagg gcatgcgcaa gccagcgttc 3360

ctgtccgggg agcagaagaa ggctatcgtg gacctcctgt tcaagaccaa ccggaaggtc 3420

acggttaagc aactcaagga ggactacttc aagaagatcg agtgcttcga ttcggtcgag 3480

atcagcggcg ttgaggaccg cttcaacgcc agcctcggga cctaccacga tctcctgaag 3540

atcatcaagg ataaggactt cctggacaac gaggagaacg aggatatcct ggaggacatc 3600

gtgctgaccc tcacgctgtt cgaggacagg gagatgatcg aggagcgcct gaagacgtac 3660

gcccatctct tcgatgacaa ggtcatgaag caactcaagc gccggagata caccggctgg 3720

gggaggctgt cccgcaagct catcaacggc atccgggaca agcagtccgg gaagaccatc 3780

ctcgacttcc tcaagagcga tggcttcgcc aacaggaact tcatgcaact gatccacgat 3840

gacagcctca ccttcaagga ggatatccaa aaggctcaag tgagcggcca gggggactcg 3900

ctgcacgagc atatcgcgaa cctcgctggc tcccccgcga tcaagaaggg catcctccag 3960

accgtgaagg ttgtggacga gctcgtgaag gtcatgggcc ggcacaagcc tgagaacatc 4020

gtcatcgaga tggccagaga gaaccaaacc acgcagaagg ggcaaaagaa ctctagggag 4080

cgcatgaagc gcatcgagga gggcatcaag gagctggggt cccaaatcct caaggagcac 4140

ccagtggaga acacccaact gcagaacgag aagctctacc tgtactacct ccagaacggc 4200

agggatatgt acgtggacca agagctggat atcaaccgcc tcagcgatta cgacgtcgat 4260

catatcgttc cccagtcttt cctgaaggat gactccatcg acaacaaggt cctcaccagg 4320

tcggacaaga accgcggcaa gtcagataac gttccatctg aggaggtcgt taagaagatg 4380

aagaactact ggaggcagct cctgaacgcc aagctgatca cgcaaaggaa gttcgacaac 4440

ctcaccaagg ctgagagagg cgggctctca gagctggaca aggccggctt catcaagcgg 4500

cagctggtcg agaccagaca aatcacgaag cacgttgcgc aaatcctcga ctctcggatg 4560

aacacgaagt acgatgagaa cgacaagctg atcagggagg ttaaggtgat caccctgaag 4620

tctaagctcg tctccgactt caggaaggat ttccagttct acaaggttcg cgagatcaac 4680

aactaccacc atgcccatga cgcttacctc aacgctgtgg tcggcaccgc tctgatcaag 4740

aagtacccaa agctggagtc cgagttcgtg tacggggact acaaggttta cgatgtgcgc 4800

aagatgatcg ccaagtcgga gcaagagatc ggcaaggcta ccgccaagta cttcttctac 4860

tcaaacatca tgaacttctt caagaccgag atcacgctgg ccaacggcga gatccggaag 4920

agaccgctca tcgagaccaa cggcgagacg ggggagatcg tgtgggacaa gggcagggat 4980

ttcgcgaccg tccgcaaggt tctctccatg ccccaggtga acatcgtcaa gaagaccgag 5040

gtccaaacgg gcgggttctc aaaggagtct atcctgccta agcggaacag cgacaagctc 5100

atcgccagaa agaaggactg ggacccaaag aagtacggcg ggttcgacag ccctaccgtg 5160

gcctactcgg tcctggttgt ggcgaaggtt gagaagggca agtccaagaa gctcaagagc 5220

gtgaaggagc tcctggggat caccatcatg gagaggtcca gcttcgagaa gaacccaatc 5280

gacttcctgg aggccaaggg ctacaaggag gtgaagaagg acctgatcat caagctcccg 5340

aagtactctc tcttcgagct ggagaacggc aggaagagaa tgctggcttc cgctggcgag 5400

ctccagaagg ggaacgagct cgcgctgcca agcaagtacg tgaacttcct ctacctggct 5460

tcccactacg agaagctcaa gggcagcccg gaggacaacg agcaaaagca gctgttcgtc 5520

gagcagcaca agcattacct cgacgagatc atcgagcaaa tctccgagtt cagcaagcgc 5580

gtgatcctcg ccgacgcgaa cctggataag gtcctctccg cctacaacaa gcaccgggac 5640

aagcccatca gagagcaagc ggagaacatc atccatctct tcaccctgac gaacctcggc 5700

gctcctgctg ctttcaagta cttcgacacc acgatcgatc ggaagagata cacctccacg 5760

aaggaggtcc tggacgcgac cctcatccac cagtcgatca ccggcctgta cgagacgagg 5820

atcgacctct cacaactcgg cggggataag agacccgcag caaccaagaa ggcagggcaa 5880

gcaaagaaga agaagacgcg tgactccggc ggcagcatgt ttggagagag ctggaagaag 5940

cacctcagcg gggagttcgg gaaaccgtat tttatcaagc taatgggatt tgttgcagaa 6000

gaaagaaagc attacactgt ttatccaccc ccacaccaag tcttcacctg gacccagatg 6060

tgtgacataa aagatgtgaa ggttgtcatc ctgggacagg atccatatca tggacctaat 6120

caagctcacg ggctctgctt tagtgttcaa aggcctgttc cgcctccgcc cagtttggag 6180

aacatttata aagagttgtc tacagacata gaggattttg ttcatcctgg ccatggagat 6240

ttatctgggt gggccaagca aggtgttctc cttctcaacg ctgtcctcac ggttcgtgcc 6300

catcaagcca actctcataa ggagcgaggc tgggagcagt tcactgatgc agttgtgtcc 6360

tggctaaatc agaactcgaa tggccttgtt ttcttgctct ggggctctta tgctcagaag 6420

aagggcagtg ccattgatag gaagcggcac catgtactac agacggctca tccctcccct 6480

ttgtcagtgt atagagggtt ctttggatgt agacactttt caaagaccaa tgagctgctg 6540

cagaagtctg gcaagaagcc cattgactgg aaggagctgt cgggggggag cccaaagaag 6600

aagcggaagg tg 6612

<210> 10

<211> 7458

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

atgccaaaga agaagaggaa ggtttcggac acgtggagct ctatccaggc ccacaagaag 60

cagctggact ctctgcggga gaggctgcag cggaggcgga agcaggactc ggggcacttg 120

gatctacgga atccagaggc agcattgtct ccaaccttcc gtagtgacag cccagtgcct 180

actgcaccca cctctggtgg ccctaagccc agcacagctt cagcagttcc tgaattagct 240

acagatcctg agttagagaa gaagttgcta caccacctct ctgatctggc cttaacattg 300

cccactgatg ctgtgtccat ctgtcttgcc atctccacgc cagatgctcc tgccactcaa 360

gatggggtag aaagcctcct gcagaagttt gcagctcagg agttgattga ggtaaagcga 420

ggtctcctac aagatgatgc acatcctact cttgtaacct atgctgacca ttccaagctc 480

tctgccatga tgggtgctgt ggcagaaaag aagggccctg gggaggtagc agggactgtc 540

acagggcaga agcggcgtgc agaacaggac tcgactacag tagctgcctt tgccagttcg 600

ttagtctctg gtctgaactc ttcagcatcg gaaccagcaa aggagccagc caagaaatca 660

aggaaacatg ctgcctcaga tgttgatctg gagatagaga gccttctgaa ccaacagtcc 720

actaaggaac aacagagcaa gaaggtcagt caggagatcc tagagctatt aaatactaca 780

acagccaagg aacaatccat tgttgaaaaa tttcgctctc gaggtcgggc ccaagtgcaa 840

gaattctgtg actatggaac caaggaggag tgcatgaaag ccagtgatgc tgatcgaccc 900

tgtcgcaagc tgcacttcag acgaattatc aataaacaca ctgatgagtc tttaggtgac 960

tgctctttcc ttaatacatg tttccacatg gatacctgca agtatgttca ctatgaaatt 1020

gatgcttgca tggattctga ggcccctggc agcaaagacc acacgccaag ccaggagctt 1080

gctcttacac agagtgtcgg aggtgattcc agtgcagacc gactcttccc acctcagtgg 1140

atctgttgtg atatccgcta cctggacgtc agtatcttgg gcaagtttgc agttgtgatg 1200

gctgacccac cctgggatat tcacatggaa ctgccctatg ggaccctgac agatgatgag 1260

atgcgcaggc tcaacatacc cgtactacag gatgatggct ttctcttcct ctgggtcaca 1320

ggcagggcca tggagttggg gagagaatgt ctaaacctct gggggtatga acgggtagat 1380

gaaattattt gggtgaagac aaatcaactg caacgcatca ttcggacagg ccgtacaggt 1440

cactggttga accatgggaa ggaacactgc ttggttggtg tcaaaggaaa tccccaaggc 1500

ttcaaccagg gtctggattg tgatgtgatc gtagctgagg ttcgttccac cagtcataaa 1560

ccagatgaaa tctatggcat gattgaaaga ctatctcctg gcactcgcaa gattgagtta 1620

tttggacgac cacacaatgt gcaacccaac tggatcaccc ttggaaacca actggatggg 1680

atccacctac tagacccaga tgtggttgca cggttcaagc aaaggtaccc agatggtatc 1740

atctctaaac ctaagaattt aagcggagga tctagcggag gatcaagcgg aagcgagact 1800

cctggaacca gcgaaagcgc aaccccagaa agcagcggag gaagtagcgg aggaagctca 1860

tcggagaccg gccctgttgc tgttgacccc accctgcggc ggagaatcga gccacacgag 1920

ttcgaggtgt tcttcgaccc aagggagctc cgcaaggaga cgtgcctcct gtacgagatc 1980

aactggggcg gcaggcactc catctggagg cacaccagcc aaaacaccaa caagcacgtg 2040

gaggtcaact tcatcgagaa gttcaccacc gagaggtact tctgcccaaa cacccgctgc 2100

tccatcacct ggttcctgtc ctggagccca tgcggcgagt gctccagggc catcaccgag 2160

ttcctcagcc gctacccaca cgtcaccctg ttcatctaca tcgccaggct ctaccaccac 2220

gccgacccaa ggaacaggca gggcctccgc gacctgatct ccagcggcgt gaccatccaa 2280

atcatgaccg agcaggagtc cggctactgc tggaggaact tcgtcaacta ctccccaagc 2340

aacgaggccc actggccaag gtacccacac ctctgggtgc gcctctacgt gctcgagctg 2400

tactgcatca tcctcggcct gccaccatgc ctcaacatcc tgaggcgcaa gcaaccacag 2460

ctgaccttct tcaccatcgc cctccaaagc tgccactacc agaggctccc accacacatc 2520

ctgtgggcta ccggcctcaa gtccggcagc gagacgccag gcacctccga gagcgctacg 2580

cctgaactta aggacaagaa gtactcgatc ggcctcgcca tcgggacgaa ctcagttggc 2640

tgggccgtga tcaccgacga gtacaaggtg ccctctaaga agttcaaggt cctggggaac 2700

accgaccgcc attccatcaa gaagaacctc atcggcgctc tcctgttcga cagcggggag 2760

accgctgagg ctacgaggct caagagaacc gctaggcgcc ggtacacgag aaggaagaac 2820

aggatctgct acctccaaga gattttctcc aacgagatgg ccaaggttga cgattcattc 2880

ttccaccgcc tggaggagtc tttcctcgtg gaggaggata agaagcacga gcggcatccc 2940

atcttcggca acatcgtgga cgaggttgcc taccacgaga agtaccctac gatctaccat 3000

ctgcggaaga agctcgtgga ctccaccgat aaggcggacc tcagactgat ctacctcgct 3060

ctggcccaca tgatcaagtt ccgcggccat ttcctgatcg agggggatct caacccagac 3120

aacagcgatg ttgacaagct gttcatccaa ctcgtgcaga cctacaacca actcttcgag 3180

gagaacccga tcaacgcctc tggcgtggac gcgaaggcta tcctgtccgc gaggctctcg 3240

aagtccagga ggctggagaa cctgatcgct cagctcccag gcgagaagaa gaacggcctg 3300

ttcgggaacc tcatcgctct cagcctgggg ctcaccccga acttcaagtc gaacttcgat 3360

ctcgctgagg acgccaagct gcaactctcc aaggacacct acgacgatga cctcgataac 3420

ctcctggccc agatcggcga tcaatacgcg gacctgttcc tcgctgccaa gaacctgtcg 3480

gacgccatcc tcctgtcaga tatcctccgc gtgaacaccg agatcacgaa ggctccactc 3540

tctgcctcca tgatcaagcg ctacgacgag caccatcagg atctgaccct cctgaaggcg 3600

ctggtccgcc aacagctccc ggagaagtac aaggagattt tcttcgatca gtcgaagaac 3660

ggctacgctg ggtacatcga cggcggggcc tcacaagagg agttctacaa gttcatcaag 3720

ccaatcctgg agaagatgga cggcacggag gagctcctgg tgaagctcaa cagggaggac 3780

ctcctgcgga agcagagaac cttcgataac ggcagcatcc cccaccaaat ccatctcggg 3840

gagctgcacg ccatcctgag aaggcaagag gacttctacc ctttcctcaa ggataaccgg 3900

gagaagatcg agaagatcct gaccttcaga atcccatact acgtcggccc tctcgcgcgg 3960

gggaactcaa gattcgcttg gatgacccgc aagtctgagg agaccatcac gccgtggaac 4020

ttcgaggagg tggtggacaa gggcgctagc gctcagtcgt tcatcgagag gatgaccaac 4080

ttcgacaaga acctgcccaa cgagaaggtg ctccctaagc actcgctcct gtacgagtac 4140

ttcaccgtct acaacgagct cacgaaggtg aagtacgtca ccgagggcat gcgcaagcca 4200

gcgttcctgt ccggggagca gaagaaggct atcgtggacc tcctgttcaa gaccaaccgg 4260

aaggtcacgg ttaagcaact caaggaggac tacttcaaga agatcgagtg cttcgattcg 4320

gtcgagatca gcggcgttga ggaccgcttc aacgccagcc tcgggaccta ccacgatctc 4380

ctgaagatca tcaaggataa ggacttcctg gacaacgagg agaacgagga tatcctggag 4440

gacatcgtgc tgaccctcac gctgttcgag gacagggaga tgatcgagga gcgcctgaag 4500

acgtacgccc atctcttcga tgacaaggtc atgaagcaac tcaagcgccg gagatacacc 4560

ggctggggga ggctgtcccg caagctcatc aacggcatcc gggacaagca gtccgggaag 4620

accatcctcg acttcctcaa gagcgatggc ttcgccaaca ggaacttcat gcaactgatc 4680

cacgatgaca gcctcacctt caaggaggat atccaaaagg ctcaagtgag cggccagggg 4740

gactcgctgc acgagcatat cgcgaacctc gctggctccc ccgcgatcaa gaagggcatc 4800

ctccagaccg tgaaggttgt ggacgagctc gtgaaggtca tgggccggca caagcctgag 4860

aacatcgtca tcgagatggc cagagagaac caaaccacgc agaaggggca aaagaactct 4920

agggagcgca tgaagcgcat cgaggagggc atcaaggagc tggggtccca aatcctcaag 4980

gagcacccag tggagaacac ccaactgcag aacgagaagc tctacctgta ctacctccag 5040

aacggcaggg atatgtacgt ggaccaagag ctggatatca accgcctcag cgattacgac 5100

gtcgatcata tcgttcccca gtctttcctg aaggatgact ccatcgacaa caaggtcctc 5160

accaggtcgg acaagaaccg cggcaagtca gataacgttc catctgagga ggtcgttaag 5220

aagatgaaga actactggag gcagctcctg aacgccaagc tgatcacgca aaggaagttc 5280

gacaacctca ccaaggctga gagaggcggg ctctcagagc tggacaaggc cggcttcatc 5340

aagcggcagc tggtcgagac cagacaaatc acgaagcacg ttgcgcaaat cctcgactct 5400

cggatgaaca cgaagtacga tgagaacgac aagctgatca gggaggttaa ggtgatcacc 5460

ctgaagtcta agctcgtctc cgacttcagg aaggatttcc agttctacaa ggttcgcgag 5520

atcaacaact accaccatgc ccatgacgct tacctcaacg ctgtggtcgg caccgctctg 5580

atcaagaagt acccaaagct ggagtccgag ttcgtgtacg gggactacaa ggtttacgat 5640

gtgcgcaaga tgatcgccaa gtcggagcaa gagatcggca aggctaccgc caagtacttc 5700

ttctactcaa acatcatgaa cttcttcaag accgagatca cgctggccaa cggcgagatc 5760

cggaagagac cgctcatcga gaccaacggc gagacggggg agatcgtgtg ggacaagggc 5820

agggatttcg cgaccgtccg caaggttctc tccatgcccc aggtgaacat cgtcaagaag 5880

accgaggtcc aaacgggcgg gttctcaaag gagtctatcc tgcctaagcg gaacagcgac 5940

aagctcatcg ccagaaagaa ggactgggac ccaaagaagt acggcgggtt cgacagccct 6000

accgtggcct actcggtcct ggttgtggcg aaggttgaga agggcaagtc caagaagctc 6060

aagagcgtga aggagctcct ggggatcacc atcatggaga ggtccagctt cgagaagaac 6120

ccaatcgact tcctggaggc caagggctac aaggaggtga agaaggacct gatcatcaag 6180

ctcccgaagt actctctctt cgagctggag aacggcagga agagaatgct ggcttccgct 6240

ggcgagctcc agaaggggaa cgagctcgcg ctgccaagca agtacgtgaa cttcctctac 6300

ctggcttccc actacgagaa gctcaagggc agcccggagg acaacgagca aaagcagctg 6360

ttcgtcgagc agcacaagca ttacctcgac gagatcatcg agcaaatctc cgagttcagc 6420

aagcgcgtga tcctcgccga cgcgaacctg gataaggtcc tctccgccta caacaagcac 6480

cgggacaagc ccatcagaga gcaagcggag aacatcatcc atctcttcac cctgacgaac 6540

ctcggcgctc ctgctgcttt caagtacttc gacaccacga tcgatcggaa gagatacacc 6600

tccacgaagg aggtcctgga cgcgaccctc atccaccagt cgatcaccgg cctgtacgag 6660

acgaggatcg acctctcaca actcggcggg gataagagac ccgcagcaac caagaaggca 6720

gggcaagcaa agaagaagaa gacgcgtgac tccggcggca gcatgtttgg agagagctgg 6780

aagaagcacc tcagcgggga gttcgggaaa ccgtatttta tcaagctaat gggatttgtt 6840

gcagaagaaa gaaagcatta cactgtttat ccacccccac accaagtctt cacctggacc 6900

cagatgtgtg acataaaaga tgtgaaggtt gtcatcctgg gacaggatcc atatcatgga 6960

cctaatcaag ctcacgggct ctgctttagt gttcaaaggc ctgttccgcc tccgcccagt 7020

ttggagaaca tttataaaga gttgtctaca gacatagagg attttgttca tcctggccat 7080

ggagatttat ctgggtgggc caagcaaggt gttctccttc tcaacgctgt cctcacggtt 7140

cgtgcccatc aagccaactc tcataaggag cgaggctggg agcagttcac tgatgcagtt 7200

gtgtcctggc taaatcagaa ctcgaatggc cttgttttct tgctctgggg ctcttatgct 7260

cagaagaagg gcagtgccat tgataggaag cggcaccatg tactacagac ggctcatccc 7320

tcccctttgt cagtgtatag agggttcttt ggatgtagac acttttcaaa gaccaatgag 7380

ctgctgcaga agtctggcaa gaagcccatt gactggaagg agctgtcggg ggggagccca 7440

aagaagaagc ggaaggtg 7458

Claims (10)

1.重组基因编辑系统，其特征在于：所述重组基因编辑系统为在基因编辑系统的基础上改造得到的，所述重组基因编辑系统表达融合蛋白，所述融合蛋白含有序列特异性结合蛋白、诱导基因组修饰因子和表观遗传因子；所述重组基因编辑系统的基因编辑效率高于所述基因编辑系统。

2.根据权利要求1所述的重组基因编辑系统，其特征在于：所述基因编辑系统为碱基编辑系统。

3.根据权利要求1或2所述的重组基因编辑系统，其特征在于：所述序列特异性结合蛋白为Cas9蛋白；所述诱导基因组修饰因子为脱氨酶。

4.根据权利要求1或2所述的重组基因编辑系统，其特征在于：所述表观遗传因子为染色质重塑因子、组蛋白修饰因子和/或RNA修饰因子。

5.根据权利要求2或3或4所述的重组基因编辑系统，其特征在于：所述基因编辑系统为CBE碱基编辑系统、GBE碱基编辑系统；所述脱氨酶为胞苷脱氨酶；所述Cas9蛋白为nCas9。

6.根据权利要求4或5所述的重组基因编辑系统，其特征在于：所述染色质重塑因子为SOX2；所述组蛋白修饰因子为SETD2；所述RNA修饰因子为METTL3；

所述SOX2为A1)、A2)、A3)、A4)或A5)中的任一种：

A1)氨基酸序列为序列表中序列1的第280-596位；

A2)由编码序列是序列表中序列6的第838-1053位的核苷酸序列编码的蛋白质；

A3)由编码序列是序列表中序列7的第838-1188位的核苷酸序列编码的蛋白质；

A4)将A1)、A2)或A3)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)、A2)或A3)衍生的或与A1)、A2)或A3)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；

A5)在A1)、A2)、A3)或A4)的N末端或/和C末端连接蛋白标签得到的融合蛋白质；

和/或，所述SETD2为B1)、B2)或B3)中的任一种：

B1)由编码序列是序列表中序列9的第25-915位的核苷酸序列编码的蛋白质；

B2)将B1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由B1)衍生的或与B1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；

B3)在B1)或B2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质；

和/或，所述METTL3为C1)、C2)或C3)中的任一种：

C1)由编码序列是序列表中序列10的第25-1761位的核苷酸序列编码的蛋白质；

C2)将C1)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由C1)衍生的或与C1)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；

C3)在C1)或C2)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

7.根据权利要求1或6所述的重组基因编辑系统，其特征在于：所述融合蛋白为D1)、D2)、D3)、D4)、D5)、D6)中的任一种：

D1)氨基酸序列为序列表中序列1的蛋白质；

D2)氨基酸序列为序列表中序列3的蛋白质；

D3)由编码序列是序列表中序列6的第1-5871位的核苷酸序列编码的蛋白质；

D4)由编码序列是序列表中序列7的第1-6006位的核苷酸序列编码的蛋白质；

D5)由编码序列是序列表中序列8的第1-6072位的核苷酸序列编码的蛋白质；

D6)由编码序列是序列表中序列9的第1-6612位的核苷酸序列编码的蛋白质；

D7)由编码序列是序列表中序列10的第1-7458位的核苷酸序列编码的蛋白质；

D8)将D1)、D2)、D3)、D4)、D5)、D6)或D7)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由D1)、D2)、D3)、D4)、D5)、D6或D7)衍生的或与D1)、D2)、D3)、D4)、D5)、D6)或D7)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；

D9)在D1)、D2)、D3)、D4)、D5)、D6)、D7)或D8)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。

8.权利要求1-7中任一权利要求中所述的融合蛋白或其相关的生物材料，所述生物材料为下述任一种：

E1)编码权利要求1-7中任一所述融合蛋白质的核酸分子；

E2)含有E1)所述核酸分子的表达盒；

E3)含有E1)所述核酸分子的重组载体、或含有E2)所述表达盒的重组载体；

E4)含有E1)所述核酸分子的重组微生物、或含有E2)所述表达盒的重组微生物、或含有E3)所述重组载体的重组微生物；

E5)含有E1)所述核酸分子的转基因植物细胞系、或含有E2)所述表达盒的转基因细胞系、或含有E3)所述重组载体的转基因细胞系；

E6)含有E1)所述核酸分子的转基因植物组织、或含有E2)所述表达盒的转基因组织、或含有E3)所述重组载体的转基因组织；

E7)含有E1)所述核酸分子的转基因动物器官、或含有E2)所述表达盒的转基因动物器官、或含有E3)所述重组载体的转基因动物器官。

9.权利要求1-7中任一权利要求中所述的表观遗传因子在提高基因编辑系统的基因编辑效率中的应用。

10.权利要求1-7中任一权利要求中所述的表观遗传因子和/或权利要求8所述的融合蛋白和/或其相关的生物材料在基因编辑中的应用。

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